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(Investigative Ophthalmology and Visual Science. 2000;41:8-15.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Expression of a Single Pair of Desmosomal Glycoproteins Renders the Corneal Epithelium Unique Amongst Stratified Epithelia

Anthea J. Messent1,2, Melanie J. Blissett2, Gillian L. Smith1, Alison J. North2, Anthony Magee3, David Foreman1, David R. Garrod2 and Mike Boulton1,4

1 From the Department of Opthalmology and the 2 Epithelial Morphogenesis Research Group, School of Biological Sciences, University of Manchester, UK; the 3 Division of Membrane Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, UK; and the 4 Cell and Molecular Biology Unit, Department of Optometry and Vision Sciences, Redwood Building, Cardiff University, Cardiff, UK.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine desmosomal glycoprotein isoform expression in bovine corneal, limbal, and conjunctival epithelium and desmosomal profile and distribution during corneal re-epithelialization.

METHODS. Immunofluorescence (IF) for desmosomal components on cryostat sections of fresh epithelia was supported by immunoblot analysis of tissue lysates. Wounded corneas maintained in organ culture were examined by IF at times up to full re-epithelialization (96 hours).

RESULTS. Immunofluorescence for desmoplakin confirmed desmosome presence throughout all three epithelia. Plakoglobin was also ubiquitous. Of the desmosomal glycoproteins, desmocollin 2 (Dsc2) and desmoglein 2 (Dsg2) were expressed throughout, but Dsc3 and Dsg3 were confined to the limbus and conjunctiva, and Dsc1 and Dsg1 were absent. Dsc2 and Dsg2 IFs were stronger in superficial layers, but Dsc3 and Dsg3 were stronger basally, fading suprabasally. Glycoprotein expression in cornea and conjunctiva was confirmed by immunoblot analysis. No change in glycoprotein expression occurred during re-epithelialization.

CONCLUSIONS. Uniquely among stratified epithelia, cornea expresses only a single pair of desmosomal glycoproteins, Dsc2 and Dsg2. Expression of Dsc3 and Dsg3 in limbus and conjunctiva coincides with their association with cell proliferation in other epithelia, but corneal epithelial cells did not express Dsc3 or Dsg3 during re-epithelialization. Absence of Dsc1 and Dsg1 correlates with lack of keratinization in ocular epithelia. These expression patterns may have significance for the specific properties and differentiation patterns of the epithelia. Presence of desmosomes throughout re-epithelialization raises the question of how migrating cells mutually re-position.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Desmosomes are intercellular, plaque-bearing adhesive junctions.1 2 3 4 5 6 The major desmosomal glycoproteins, the desmocollins (Dsc) and desmogleins (Dsg), are members of the cadherin family of cell adhesion molecules. Three distinct isoforms of both desmocollin (Dsc1 through Dsc3) and desmoglein (Dsg1 through Dsg3) have been isolated, each protein the product of a separate gene.6 7 8 Other desmosomal proteins include plakoglobin, plakophilin, and desmoplakin, which bind to desmosomal glycoproteins in the plaque region.9 10 11 12 13 Desmoplakin links desmosomes to the cytoskeleton.14 15 16 17 18 19

Dsg2 and Dsc2 have been detected, at least at the mRNA level, in all desmosome-containing tissues, including simple epithelia such as colon, small intestine, and non-epithelial tissue such as myocardium.20 21 However, many stratified epithelia such as tonsil and esophagus have been found to express Dsc3 and Dsg3,13 14 along with Dsc2 and Dsg2. Epidermis, a cornified stratified squamous epithelia, expresses Dsc1 and Dsg1 in addition to Dsc2, Dsg2, Dsc3, and Dsg3.22 23 24 25 26 Dsc1 and Dsg1 are also expressed in the papillae of tongue epithelium23 26 In the epidermis, the desmosomal glycoproteins are differentially distributed through the epithelial layers. Dsc1 is most strongly expressed suprabasally in the spinous layer of epidermis, weakly expressed in the juxta-basal layers, and absent from the basal cell layer. In contrast, Dsc3 is most strongly expressed in the epidermal basal layer and Dsc2 in the first few suprabasal layers in the bases of the rete ridges.23

Distribution of Dsc1 and Dsc3 in the epidermis may be related to the differentiation state of cells within the epithelial layer. A reciprocal grading of immunostaining intensity for Dsc1 and Dsc3 has been described in bovine nasal epidermis,28 suggesting that desmosomal glycoprotein expression is modulated during epidermal cell differentiation and progression toward the cornified layer. The patterns of Dsg1 and Dsg3 expression resemble those of the corresponding Dsc isoforms, whereas Dsg2 expression is most strongly associated with the basal layer.29 30

Although the desmosomal profile of tissues such as the epidermis has been well characterized, little is known about the stratified, non-cornified corneal epithelium. Desmosomes are known to be present throughout the corneal epithelial cell layers,31 particularly between the interdigitating cell borders of wing cells,32 33 but the desmosomal glycoprotein isoform distribution within these junctions has yet to be elucidated.

We have used fresh bovine tissue to determine the expression of specific desmosomal glycoproteins within the different cell layers of corneal, limbal, and conjunctival epithelia. We also investigated whether desmosomal junctions were retained during the considerable organizational changes that occur to the epithelial layer during corneal re-epithelialization. Our results show that the corneal epithelium is unique among stratified epithelia in possessing a single pair of desmosomal glycoproteins and indicate that desmosomes are important in maintaining the integrity of the cell sheet during corneal re-epithelialization after the wounding.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue
Bovine eyes and muzzle were obtained from Newton Heath abattoir, Manchester, UK, within 2 hours of slaughter and maintained on ice. A sample of human foreskin, obtained within 24 hours of surgery, was provided by the Hope Hospital, Manchester, UK.

Organ Culture
Corneal organ culture of both wounded and unwounded bovine corneas was undertaken as previously described by Foreman et al.34 Eyes were disinfected by a brief immersion in 20% povidine–iodine solution (Betadine; Seton Healthcare Group, Oldham, UK), then rinsed with sterile phosphate-buffered saline (PBS). A 5-mm excisional trephine was used to create a single wound in the center of each eye, penetrating to approximately one third of the depth of the cornea. Corneoscleral rims from both wounded and unwounded eyes (controls) were excised and placed in culture. After 20 minutes and 1, 3, 6, 12, 24, 36, 48, and 96 hours in culture, one unwounded cornea and two wounded corneas per experiment were removed from culture and bisected through the middle of the cornea or the wound area. This experiment was repeated on at least three separate occasions. Excess sclera was trimmed away and the corneas embedded directly in OCT compound (Tissue-Tek; Agar Scientific, Stanstead, UK) over liquid nitrogen. Frozen blocks were stored at –20°C and 5.0 µm sections cut using a Kryostat 1720 (Leica, Milton Keynes, UK). Sections were stored at –70°C until use.

Antibodies
The primary antibodies used were JCMC (rabbit polyclonal anti-Dsc1),28 ED-E (guinea pig polyclonal anti-Dsc2, described below), 07-4G (mouse monoclonal anti-Dsc3),25 33-3D (mouse monoclonal anti-Dsg2),35 and 11-5F (mouse monoclonal anti-desmoplakin).36 P23 (mouse monoclonal anti-Dsg1)37 was purchased from Insight Biotechnology (Wembley, UK). 11-E4 (mouse monoclonal anti-plakoglobin)38 was the kind gift of Margaret Wheelock, (Department of Biology, University of Toledo, Ohio). Antiserum number 10 (No.10; rabbit polyclonal anti-Dsg3) is described below.

The secondary antibodies used were dichlorotriazinylamino fluorescein (DTAF)–conjugated donkey anti-mouse IgG, fluorescein isothiocyanate (FITC)–conjugated donkey anti-mouse IgM, FITC-conjugated donkey anti-rabbit IgG, and FITC-conjugated donkey anti-guinea pig IgG (Jackson Laboratories, West Grove, PA).

Preparation of Polyclonal Dsc2- and Dsg3-Specific Antibodies
Polyclonal antibody against Dsc2 was generated as follows. An expression vector, pGEX-4T-3Dsc2, encoding a 600-bp fragment of extracellular domains 4 to 5 (EC4–5) of murine Dsc2 linked to the glutathione S-transferase gene was generated using a cDNA obtained after screening of an 8.5-day mouse embryo cDNA library.39 The 600-bp fragment was subcloned into the pGEX-4T-3 vector (Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK), sequenced, and transformed into JM101 bacterial cells. Using the glutathione S-transferase (GST) gene fusion system (Amersham Pharmacia Biotech), the cDNA clone was grown and purified as per the manufacturer’s protocol. The resulting Dsc2/GST fusion protein was mixed with TitreMax Gold adjuvant (CytRx Corporation/Stratech Scientific Ltd., Luton, UK) as per manufacturers protocol and injected into guinea pigs. A booster was given 4 weeks later and the animal terminally bled after another 4 weeks.

The resultant antibody was affinity purified on two cyanogen bromide–activated Sepharose-4B columns coupled to the Dsc2/GST fusion protein and coupled to the GST fusion protein, then concentrated. Vectors encoding full-length Dsc1b (pGEX-3X/Dsc1b), Dsc2b (pGEX-4T/Dsc2b), and Dsc3b (pGEX-2T/Dsc3b) linked to GST were generated and used for immunoblot analysis as previously described.28 Immunolabeling of bovine nasal epidermis resulted in a staining pattern that was different from Dsc1 or Dsc3, but some weak cross-reactivity of the antibody with Dsc3 was detected on immunoblots. Although it is possible that the antibody only recognizes Dsc3 under denaturing conditions, we cannot at present exclude the possibility that it binds with low affinity to the native form of Dsc3.

Polyclonal antiserum No.10 was raised against a synthetic peptide corresponding to the last 11 amino acids of the C terminus of human Dsg3 (LCTEDPCSRLI in one letter amino acid code). The peptide, produced by standard Fmoc synthesis, was coupled to maleimide-activated keyhole limpet hemocyanin (Pierce Europe; Oud Beijerland, The Netherlands) and injected into rabbits. After several booster injections of the peptide antigen, antibody specific to Dsg3 was affinity purified from immunized rabbit serum diluted 1:1 with PBS, by adsorption to the immunizing peptide coupled to NHS-activated HiTrap columns (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.

The specificity of the resulting antibody was verified by immunoblot analysis against lysates from cells expressing a known complement of desmogleins. The antibody failed to recognize bands in the cell lysates at the known relative molecular weight of Dsg1 or Dsg2, reacting with a single band at the known molecular weight of Dsg3. The antibody also demonstrated some species specificity, recognizing human and bovine Dsg3 but not mouse or dog Dsg3.

All animals used in the preparation of antibodies were housed and treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Immunohistochemistry
Frozen sections were allowed to reach room temperature and treated with 0.05% Triton X-100 (Sigma, St. Louis, MO) in PBS for 30 minutes to permeabilize the tissue. The sections were blocked in 5% normal goat serum (Sigma Aldrich Co., Poole, UK) and 2% bovine serum albumin (BSA; Sigma Aldrich) in PBS for 30 minutes. Sections were washed in 0.25% BSA in PBS (wash solution) and incubated with primary antibodies for 1 hour in wash solution. Unbound primary antibody was removed by several changes of wash solution. Secondary antibodies conjugated to FITC (Jackson Laboratories) were used at 1:100 dilution in wash solution and incubated with sections for 30 minutes. Excess secondary antibody was washed off with several changes of wash buffer and sections mounted using Gelvatol (Fisons, Loughborough, UK).

Sections were examined using an Axiophot fluorescence microscope (Zeis, Oberkochen, Germany). Sections of cryopreserved bovine nasal epidermis (prepared in the same manner as bovine cornea) were used as a positive control for antibody staining, because the molecular structure of desmosomes in this tissue has been well characterized (see Ref. 2 and references therein). Purified mouse or guinea-pig immunoglobulins (Sigma Aldrich) were used as a negative control in place of the respective primary antibodies where the Ig concentration was known. Otherwise, controls were taken from sections incubated with secondary antibody alone.

Western Blot Analysis
Conjunctival and central bovine corneal epithelial sheets were isolated from unwounded eyes by incubating excised tissue in Dispase II solution (2.4 U/ml; Boehringer Mannheim, Mannheim, Germany) for 2 hours at 37°C. The epithelial layer was gently separated from the stroma and homogenized in 2x sample buffer (Bio-Rad, Hemel Hempstead, UK) with 5% ß-mercaptoethanol (reducing sample buffer). Samples were heated to 100°C for 5 minutes and stored at -70°C.

Bovine nasal and human foreskin epidermis was dissected from the tissue and snap-frozen in liquid nitrogen. Frozen epidermis was ground to powder and homogenized in reducing sample buffer. Samples were boiled and stored as for the corneal and conjunctival epithelial preparations.

Bovine nasal epidermis, which expresses all three Dsc and Dsg isoforms, was used as a positive control for antibodies that were known to react with bovine tissue. In addition, human foreskin epidermis was used as a positive control for P23, because this antibody had not previously been tested on bovine tissue (personal communication, Insight Biotechnology, Wembley, UK). Because the nasal epidermal lysate contained considerably more desmosomal protein than either the corneal or conjunctival epithelial lysates (as determined by Coomassie blue staining of sodium dodecyl sulfate gels), at least twice as much corneal and conjunctival lysates were loaded per track of each gel compared with nasal lysate.

Western blot analysis was performed essentially as previously described.40 Samples were separated by 4% to 10% gradient sodium dodecyl sulfate–polyacrylalmide gel electrophoresis and transferred onto Hybond-C nitrocellulose membrane (Amersham Pharmacia Biotech). Unbound membrane was blocked with 5% nonfat milk solution and incubated in the primary antibodies for 1 hour at room temperature. After extensive washing, blots were incubated for 1 hour in horseradish peroxidase–conjugated secondary antibodies, and bound protein was detected using enhanced chemiluminescence (ECL) reagents (Amersham Pharmacia Biotech).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of Desmosomal Proteins in Fresh Bovine Corneal and Conjunctival Epithelia as Determined by Western Blot Analysis
No Dsc1 was detected in bovine corneal epithelial lysate by Western blot analysis, although both the Dsc1 "a" form and "b" form were recognized in the nasal epidermal lysate. Some weak reactivity of the Dsc1 antibody (JCMC) was seen with bands of varied mobility in conjunctival epithelial lysate, but this was considered to be nonspecific (Fig. 1A ). Two bands were recognized by the Dsc2 antibody (ED-E) in nasal, corneal, and conjunctival lysates (Fig. 1B) . These bands, which correspond to the Dsc2 "a" and "b" splice variants, consistently showed slightly greater electrophoretic mobility in the corneal lysate than in the nasal or conjunctival lysates, suggesting some difference in post-translational modification in the cornea.



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  		Figure 1. Western blot analysis of bovine nasal (N), corneal (C), and conjunctival (Conj) epithelia with antibodies to the major desmosomal proteins. Human foreskin lysate (Sk) was used as the positive control for the Dsg1 antibody. (A) Anti-Dsc1 antibody (JCMC) recognized two bands in the nasal lysate but did not react with any desmosomal proteins in the corneal or conjunctival lysates. (B) Anti-Dsc2 (ED-E) antibody recognized two bands in all three bovine tissue lysates, although the doublet in the corneal lysate had greater electrophoretic mobility than the doublets in the nasal or conjunctival lysates. (C) Dsc3 antibody (07-4G) did not react with corneal lysate but did react with a doublet in the nasal lysate. (D) The major band of approximately 160 kDa, which was recognized by the anti-Dsg1 antibody (P23), was found in both the human skin and the bovine nasal lysate. However, this band was completely absent from both the corneal and conjunctival lysates. (E) Anti-Dsg2 antibody (33-3D) recognized a major band of approximately 160 kDa in all three bovine tissues, corresponding to Dsg2. (F) Anti-Dsg3 antibody (antiserum No.10) recognized a single band in the nasal and conjunctival lysate but no proteins in the corneal lysate. (G) Anti-desmoplakin antibody (11-5F) reacted with a protein doublet in all three bovine tissue lysates. (H) Anti-plakoglobin antibody (11-E4) also reacted with all three bovine tissue lysates, recognizing a single band.

 
The Dsc3 antibody (07-4G) reacted strongly with bovine nasal lysate but not at all with bovine corneal lysate (Fig. 1C) . We were unable to find any reactivity of the Dsc3 antibody to the conjunctival lysate, even though gels were maximally loaded and the conjunctival epithelium stained strongly with this antibody (see below).

The major band recognized by the anti-Dsg1 antibody (P23) in the human epidermal lysate and bovine nasal lysate corresponded to the expected relative mobility of Dsg1, approximately 160 kDa. This band was not found in either the corneal or the conjunctival lysates, suggesting that Dsg1 is absent from bovine corneal and conjunctival epithelia (Fig. 1D) . In contrast, the Dsg2 antibody reacted with all three bovine epithelial lysates (Fig. 1E) , recognizing a somewhat diffuse band at 160 kDa, the expected relative mobility of Dsg2. The low-molecular-weight proteins detected by the monoclonal anti-Dsg1 antibody at 66 kDa and the anti-Dsg2 antibody at 80kDa may be degradation products of Dsg1 and Dsg2 generated by proteolytic activity during sample preparation,35 or may simply reflect nonspecific reactivity of the antibodies due to the high protein loading of these gels.

The Dsg3 antibody (antiserum No.10) reacted with a single band in the bovine nasal lysate and in the conjunctival lysate (Fig. 1F) . No band was recognized in the corneal lysate, indicating that Dsg3 is absent from bovine corneal epithelium.

All three bovine epithelial lysates reacted strongly with both the anti-desmoplakin antibody (11-5F) and the anti-plakoglobin antibody (11-E4; Figs. 1F 1G ), recognizing bands at the expected relative mobility for the respective desmosomal proteins (250 kDa for desmoplakin 1, 215 kDa for desmoplakin 2, and 83 kDa for plakoglobin).

Expression and Distribution of Desmosomal Proteins in Fresh Bovine Ocular Epithelia as Determined by Immunofluorescence
Fresh corneas displayed a distinct staining pattern for desmosomal proteins in the corneal, limbal, and conjunctival epithelia. All three epithelia expressed desmoplakin (Figs. 2A 2C 2E ) and plakoglobin (Figs. 2B 2D 2F ). Immunostaining for desmoplakin and plakoglobin was most prominent in the superficial and mid layers of the corneal and limbal epithelium but was more evenly distributed throughout the conjunctival epithelium.



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  		Figure 2. Cryosections of normal bovine cornea, limbus, and conjunctiva were stained with anti-desmoplakin and plakoglobin antibodies. Anti-desmoplakin antibody stains throughout the corneal (A), limbal (C), and conjunctival (E) epithelia. Anti-plakoglobin antibody also stains throughout the corneal (B), limbal (D), and conjunctival (F) epithelia. Control sections were stained with just the secondary antibody alone (insets of A and B). Scale bars, 50 µm. In each case, the basal surface is indicated with an arrow.

 
Dsc1 was not detected in any of the bovine epithelia by probing with a specific anti-Dsc1 antibody (JCMC). This is consistent with the data from Western blot analysis of the corneal and conjunctival lysates. In addition, no immunostaining for Dsg1 could be detected in the cornea, limbus, or conjunctiva using the Dsg1-specific antibody (P23).

Immunofluorescence for Dsc2 and Dsg2 in corneal epithelium was graded from high intensity in the superficial cell layer to sparse punctate staining in the basal layer (Figs. 3A 3B ). Dsc2 and Dsg2 expression was more evenly distributed throughout the limbus and conjunctiva (Figs. 3C 3D 3E 3F ), with the exception of the limbal basal layer where Dsg2 could hardly be detected (Fig. 3D) .



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  		Figure 3. Cryosections of normal bovine cornea, limbus, and conjunctiva were stained with antibodies directed against Dsc2 and Dsg2. Anti-Dsc2 (D and E) antibody appeared to stain the superficial and suprabasal cells more prominently than the basal cells of the corneal (A) and limbal (C) epithelia, although staining for Dsc2 was more evenly distributed throughout the conjunctival epithelium (E). Anti-Dsg2 antibody (33-3D) stains all cell layers of the corneal (B), limbal (D), and conjunctival (F) epithelia. Control sections were stained with purified immunoglobulin in place of the primary antibody (inset of A) or secondary antibody alone (inset of B). Scale bars, 50 µm. The basal surface is indicated by an arrow.

 
Desmocollin 3 was not detected in the cornea (Fig. 4A ), but was strongly expressed by basal cells and mid-region cells of the limbal epithelium (Fig. 4C) . Dsc3 was strongly expressed in the basal cells of the conjunctival epithelium, fading in the suprabasal layer (Fig. 4E) . The expression pattern of Dsc3 in the cornea, limbus, and conjunctiva was mirrored by the expression pattern of Dsg3, which was completely undetected in the cornea, strongly expressed by the basal and mid-region cells of the limbus, and graded in expression from the basal cells in the conjunctiva (Figs. 4B 4D 4F ).



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  		Figure 4. Cryosections of normal bovine cornea, limbus and conjunctiva were stained with antibodies directed against Dsc3 (07-49) and Dsg3 (No. 10). Anti-Dsc3 antibody failed to stain any cell layers in the corneal epithelium (A). Most limbal cells proximal to the conjunctiva, apart from the superficial layer, stained prominently with the anti-Dsc3 antibody (C), although staining became progressively less intense near the limbal-corneal interface (not shown). Dsc3 staining in the conjunctiva was strong in the basal cells and gradually decreased in the suprabasal cells (E). The anti-Dsg3 antibody staining patterns of the cornea, limbus, and conjunctiva exactly mirrored those for anti-Dsc3. No staining for Dsg3 was found in the corneal epithelium (B). Basal and suprabasal cells of the limbal epithelium proximal to the limbal-corneal interface stained with the anti-Dsg3 antibody (D). Conjunctival staining with the anti-Dsg3 antibody decreased from the basal layer upward (F). Control sections were stained with secondary antibody alone (inset, A) or purified immunoglobulin in place of the primary antibody (inset, B). Scale bars, 50 µm. Basal surface indicated by an arrow.

 
Expression and Distribution of Desmosomal Proteins in the Bovine Cornea During Re-epithelialization as Determined by Immunofluorescence
Re-epithelialization of 5-mm corneal wounds was complete in organ culture by 72 hours. The morphology of the epithelial cells at the wound edge appeared to be modified during the healing process. Initially, the wound edge retained a stratified epithelial appearance. The basal, suprabasal, and superficial layers of the epithelium were clearly discernible up to 6 hours after wounding (data not shown). However, by 24 hours after wounding, basal cells of columnar morphology were absent from the leading edge of the wound, and the staining of the superficial cell layer was greatly diminished in intensity (Fig. 5C ). By 36 hours, just before wound closure, the leading edge of the modified epithelial layer had thinned to 4 to 5 cells in depth, with all cells having a flatter morphology (Fig. 5D) . After wound closure the epithelium re-stratified to full thickness (8–10 cells in depth), and the columnar basal layer was restored (Fig. 5E) .



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  		Figure 5. The staining with anti-desmoplakin antibody (11-5F) during the corneal re-epithelialization process. All epithelial cells stained for desmoplakin at all stages throughout wound healing. At time 0 (A), the pattern of desmoplakin staining resembled that seen with the glycoprotein antibodies shown in Figures 2 3 and 4 , being strongest in the superficial layers. This pattern was unchanged 3 hours after wounding (B). By 24 hours after wounding, the wound edge had become rounded and the pattern of staining changed, being most intense in the mid-region (C) of the tip (arrow). By 36 hours after wounding the wound edge had become thin and stained uniformly for desmoplakin (D). After wound closure, re-stratification and re-establishment of the initial staining pattern were completed by 48 hours (E). (F) shows a negative control section. Scale bar, 50 µm. Where appropriate the basal surface is indicated by a thick arrow and the wound edge by a thin arrow.

 
The expression pattern of the desmosomal proteins at the wound edge during wound healing closely resembled that observed for unwounded controls. Immunofluorescence for plakoglobin and desmoplakin was intense in the superficial layers, gradually decreasing in intensity through the mid layers to the basal layer before and immediately after wounding, and after wound closure (desmoplakin data Figs. 5A 5B and 5E ; data for plakoglobin not shown). By 24 hours after wounding, when no cells of basal morphology were present at the wound edge, staining for desmoplakin and plakoglobin was most intense in the central region of the wound tip, with the intense staining of the superficial layer having diminished (Fig. 5C ; data for plakoglobin not shown). By 36 hours after wounding, the greatly thinned epithelium at the wound edge showed uniformly bright staining, even in cells at the extreme tip (Fig. 5D) . Control sections showed no immunofluorescence (Fig. 5F) .

Wounded corneas remained positive for Dsc2 and Dsg2 throughout the wound healing process. At early time points and after wound closure, strongest staining for Dsc2 and Dsg2 was observed in the superficial cell layers, becoming less intense toward the basal cell layer (Figs. 6A 6B 6E ). These patterns resembled those seen in unwounded controls. At 24 and 36 hours after wounding the intense staining of superficial layer of cells was lost, and staining for Dsc2 and Dsg2 became much more evenly distributed between the cells layers (Figs. 6C and 6D ; data for Dsc2 not shown).



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  		Figure 6. All cell layers of the wounded corneal epithelium stained with anti-Dsc2 (ED-E) and anti-Dsg2 (33-3D) antibodies at each time point. Staining with anti-Dsg2 antibody in the stratified wound edge at time 0 (A), 3 hours post wounding (B), and after re-stratification (E) was more prominent in the superficial and mid-layers of the epithelium. Staining of the rounded wound tip at 24 hours post-wounding (C), and the flattened wound tip at 36 hours after wounding (D), was distributed evenly throughout all cell layers. Immunostaining for Dsc2 exactly mirrored the pattern observed for Dsg2 (not shown). Control sections treated with secondary antibody alone showed no immunofluorescence (F). Scale bars, 50 µm. Where appropriate the basal surface is indicated with a thick arrow and the wound edge with a thin arrow.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The cornea appears to be unique among complex stratified epithelia in expressing just one pair of desmosomal glycoproteins, Dsc2 and Dsg2. All other such epithelia examined so far express two or three Dsc and Dsg isoforms (Table 1) . This result shows that expression of multiple desmosomal glycoproteins is not essential for epithelial stratification.


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  		Table 1. Distribution and Intensity of Expression of the Desmosomal Glycoproteins in the Epithelial Cell Layers of Bovine Skin, Cornea, and Conjunctiva

 
In most other stratified epithelia, Dsc3 and Dsg3 are strongly expressed in the basal layer, their expression declining gradually in suprabasal layers. They are thus associated with, although clearly not confined to, the cell layers that contain stem cells. It is intriguing, therefore, to find that these isoforms are absent from the cornea itself but that they are expressed in the limbus where these corneal stem cells reside. As in other epithelia, they are not confined to the stem cell layer but extend to suprabasal layers. In the conjunctiva, Dsc3 and Dsg3 expression declines suprabasally in a pattern closely resembling that found in the epidermis. This strong association of Dsc3 with the basal layer of epidermis arises during development at the time when the adult pattern of epidermal differentiation is established (embryonic day 15) in the mouse.41 Before that stage, Dsc3 is expressed in suprabasal layers. It will be interesting to study development of the corneal epithelium and limbus to determine whether wider early expression occurs here or whether Dsc3 is confined to the limbus from the onset of its expression. Furthermore, it will be interesting to study the expression of desmosomal glycoproteins in disease. For example, it is likely that the epithelial cell fibrovascular outgrowths known as pterygia express Dsc2/Dsg2 and Dsc3/Dsg3, because they are believed to originate from the conjunctival epithelium.

Dsc1 and Dsg1 are completely absent from the ocular epithelia. This is probably related to the absence of cornification from the eye, because these isoforms are associated with terminal differentiation, leading to cornification in the epidermis. It will be interesting to discover whether these isoforms are re-expressed in cornifying diseases of the eye, such as cicatricial pemphigoid.

Absence of Dsc1/Dsg1 from the cornea and confinement of Dsc3/Dsg3 to the limbus are presumably in some way related to the special clarity required in the corneal epithelium. The eyelid provides a cornified protective layer that can be moved into place when protection is required. Although not cornified, the ocular epithelia require strong intercellular adhesion, especially between their superficial cells, to maintain the integrity of the epithelia against the mild but persistent abrasion that occurs during blinking and eye movements. It has been reported that desmosomes are most numerous in these superficial layers.42 This is consistent with the most intense immunofluorescence for desmosomal components being found in these layers. Ultrastructural evidence showing fewer desmosomes in the basal layer is also consistent in the generally weaker staining for these components found in the basal layer.42

Although essential in the normal cornea, such strong adhesion in the superficial layers is presumably less compatible with the cell movements required for re-epithelialization of corneal wounds. It is interesting, therefore, that a consistent change in desmosomal expression found during wound healing was reduction of intense immunofluorescence in the superficial layers of the advancing epithelium. This may indicate either that superficial layers do not participate in re-epithelialization or that there is a reduction of desmosome expression in these layers, presumably to facilitate cell motility. Desmosome expression clearly persists in all cells at the wound edge.43 This suggests that desmosomes, like adherens junctions, are important in maintaining the integrity of the migrating cell sheet during re-epithelialization. However, this persistence of desmosomes still poses the question of how mutual repositioning of cells during wound closure takes place. We suggest that there must be some transient modulation of cell-cell adhesion to allow cells to break and reform their contacts.

Before beginning this study, we entertained the possibility that re-epithelialization might involve a change in the pattern of desmosomal glycoprotein isoforms. This expression of Dsc3 and Dsg3 might extend into the cornea because the population of transit amplifying cells in the basal layer increased to replace lost cells by centripetal influx. No such change in Dsc/Dsg isoform expression was found. This result is perhaps consistent with the finding that the highest level of expression of Dsc2, the isoform expressed by the cornea, was in the transit amplifying cell regions of the rete ridges of the epidermis.

In conclusion, the ocular epithelia show a unique and specialized pattern of desmosomal glycoprotein expression that is consistent with their specialized functions.


   Acknowledgements
 
We thank Brian Trinnaman for help with production and characterization of antiserum No. 10.


   Footnotes
 
Supported by the Grant 051571, Wellcome Trust, UK.

Submitted for publication April 23, 1999; revised July 19, 1999; accepted August 6, 1999.

Commercial relationships policy: N.

Corresponding author: Mike Boulton, Cell and Molecular Biology Unit, Department of Optometry and Vision Sciences, Redwood Building, Cardiff University, P.O. Box 905, Cardiff, CF1 3XF, UK. boultonm{at}cardiff.ac.uk


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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