1 Laboratory of Molecular and Developmental Biology, National Eye Institute,
National Institutes of Health, Bethesda, MD 20892-2730, USA
2 Department of Biological Sciences, University of Delaware, Newark, DE 19716,
USA
3 Laboratory of Mechanisms of Ocular Disease, National Eye Institute, National
Institutes of Health, Bethesda, MD 20892-2735, USA
* Author for correspondence (e-mail: joramp{at}nei.nih.gov)
Accepted 17 February 2003
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Summary |
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Key words: Pax6, Cornea, Adhesion, SEY mice, Development
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Introduction |
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The causal factors that orchestrate corneal morphogenesis and continued
turnover of the adult stratified epithelium have not been elucidated. Here, we
have explored the role of Pax6 in corneal development. Pax6 is a
member of the Pax gene transcription factor family
(Walther and Gruss, 1991;
Walther et al., 1991
).
Heterozygous mutations in Pax6 result in a spectrum of eye
abnormalities in humans, including aniridia, Peter's anomaly and autosomal
dominant keratitis (Glaser et al.,
1990
; Glaser et al.,
1992
; Hanson et al.,
1994
), and a distinct small eye syndrome in the Small eye (SEY)
mouse and rat (Hill et al.,
1991
; Matsuo et al.,
1993
). SEY mice also have defects in parts of the central nervous
system (Callaerts et al.,
1997
; Glaser et al.,
1994
; Tremblay and Gruss,
1994
). In mice, Pax6Sey,
Pax6SeyNeu and Pax6Coop represent
three SEY strains with different point mutations in Pax6 whereas
Pax6SeyDey and Pax6SeyH strains carry
Pax6 gene deletions (Favor et
al., 1988
; Hill et al.,
1991
; Hogan et al.,
1986
; Lyon et al.,
1979
; Lyon et al.,
2000
; Schmahl et al.,
1993
; Theiler et al.,
1978
; Theiler et al.,
1980
). Homozygous SEY mice lack eyes and olfactory structures and
die at birth (Grindley et al.,
1995
; Hogan et al.,
1986
). The semidominant heterozygous phenotypes from different SEY
strains of mice show comparable developmental ocular abnormalities, including
defects in eye size, lens and retina, although the severity of the phenotypes
can vary (Callaerts et al.,
1997
; Hill et al.,
1991
). Cataracts, glaucoma and corneal opacities can develop in
mutant SEY mice after birth (Lyon et al.,
2000
; Theiler et al.,
1978
). Phenotypic variability is observed within a single SEY
strain (Hogan et al., 1986
;
Theiler et al., 1978
), even
between two eyes of the same mouse, and might result from a stringent
requirement that Pax6 activity be present at specific levels at precise times
during development (Hill et al.,
1991
; Schedl et al.,
1996
; van Raamsdonk and
Tilghman, 2000
). Recent functional studies of Pax6 using
transgenic technology have revealed specific requirements for Pax6 during lens
and retinal development (Ashery-Padan and
Gruss, 2001
). Moreover, Pax6(5a), one of the two major Pax6
isoforms, is required postnatally to establish various aspects of a normal
iris, lens, retina and cornea (Singh et
al., 2002
).
The expression pattern of Pax6 coincides well with the phenotypes
observed in SEY mice. Pax6 is expressed at mouse embryonic day 8 in
expanded regions of surface and neural ectoderm
(Walther and Gruss, 1991).
Subsequently, expression is restricted to lens and olfactory placodes,
forebrain, hindbrain, neural tube and optic vesicle
(Grindley et al., 1995
;
Walther and Gruss, 1991
). At
mid-gestation, Pax6 protein is detected in most cells of the neural retina and
all parts of the eye derived from surface ectoderm, including the lens pit,
lens vesicle, lens and cornea (Davis and
Reed, 1996
). At six weeks of age, Pax6 is restricted to a subset
of retinal neurons, the lens, cornea, conjunctiva, iris and ciliary body
(Davis and Reed, 1996
;
Koroma et al., 1997
),
consistent with a function in the adult as well as in the embryonic eye.
Some downstream molecular targets mediating the effects of Pax6 activity in
the eye have been identified. The expression of several transcription factors
in developing lens and retina is linked to Pax6
(Ashery-Padan et al., 2000;
Foerst-Potts and Sadler, 1997
;
Grindley et al., 1995
;
Marquardt et al., 2001
;
Sakai et al., 2001
;
Xu et al., 1997
). Pax6 acts as
a positive and negative regulator of crystallin gene expression
(Chambers et al., 1995
;
Cvekl et al., 1994
;
Cvekl et al., 1995
;
Duncan et al., 1998
;
Gopal-Srivastava et al., 1996
;
Richardson et al., 1995
). Pax6
has been shown to activate the corneal gene promoters for keratin 12
(K12) (Liu et al.,
1999
; Shiraishi et al.,
1998
) and gelatinase B
(Sivak et al., 2000
). Recent
microarray analysis identified many genes that were expressed differently when
Pax6 was misexpressed in the eye
(Chauhan et al., 2002
). Of
particular significance to this study, overexpression of Pax6(5a) in
transgenic mice resulted in an increase in lens expression of
5 and
ß1 integrins, paxillin, and p120ctn
(Duncan et al., 2000
), whereas
a reduction in N-cadherin expression was found in cells of the developing lens
in the SEY (+/) mouse (van
Raamsdonk and Tilghman, 2000
). Here, we show that two strains of
SEY mice have abnormal corneal morphology, especially in the epithelium.
Furthermore, we present evidence supporting the idea that alterations in cell
adhesion contribute to the mutant corneal phenotype. Importantly, our results
suggest that Pax6 is required not only for embryonic development but also for
the postnatal (PN) development and maintenance of the adult cornea.
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Materials and Methods |
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Histology
Whole eyes were immersion fixed in 4% paraformaldehyde in
phosphate-buffered saline (PBS) overnight at 4°C, washed in PBS and then
in saline for 30 minutes each, dehydrated through a series of ethanol washes,
cleared in xylene and embedded in paraffin. Prior to staining, 10 µm
sections were deparaffinized, rehydrated using a graded series of ethanol
washes and rinsed in distilled water for 15 minutes. Eye sections were stained
in Gill's hematoxylin and eosin as described
(Luna, 1968).
Electron microscopy
Whole eyes were removed, slit at the limbus, fixed for 24 hours or more at
room temperature in a solution of 2.5% glutaraldehyde and 6% sucrose buffered
to pH 7.2 with 50 mM sodium cacodylate. Small (0.5x1 mm) portions of the
central cornea were processed for electron microscopy by dehydration in an
ethanol series and embedding in epoxy resin. Ultrathin sections were stained
with uranyl acetate and lead citrate, then examined and photographed using a
JEM-100CX electron microscope (JEOL, Peabody, MA).
Immunohistochemistry
Fresh-frozen eye sections (10 µm) were collected on Superfrost/Plus
slides (Fisher, Pittsburgh, PA) and stored at 80°C until further
use. All steps were carried out at room temperature except where noted
otherwise. Tissue sections were fixed in 4% paraformaldehyde in PBS for 10
minutes and processed as reported previously
(Davis and Reed, 1996). For
fluorescent microscopy, the sections were incubated with rhodamine
(TRITC)-conjugated AffiniPure F(ab')2 Fragment Donkey
anti-mouse or anti-rabbit (Jackson ImmunoResearch Laboratories, West Grove,
PA) according to the manufacturer's instructions. Slides were counterstained
with DAPI as described (Davis and Reed,
1996
), coverslipped using Aqua-Poly/Mount (Polysciences,
Warrington, PA) and photographed using a Zeiss Axioplan2 microscope with Spot
camera.
The following antibodies were used: anti-Pax6 (1:200; a gift from R. Reed,
Johns Hopkins University, Baltimore, MD); anti-N-terminal and anti-C-terminal
K12 (1 µg ml1; a gift from W. Kao, University of
Cincinnati, Cincinnati, OH); cytokeratin 4 clone 6B10 (1:10, ICN Biomedicals,
Aurora, OH); anti-laminin (1:50; Sigma, St Louis, MO); anti-ZO-1 and
anti-E-cadherin (1 µg ml1 each; Zymed Laboratories, San
Francisco, CA); anti-5,
6, ß1 and ß4 integrins (0.5
µg ml1 each; BD PharMingen, San Diego, CA);
anti-epidermal growth factor receptor (EGFR) and anti-ErbB2 (1:200 each; Santa
Cruz Biotechnology, Santa Cruz, CA).
Phalloidin staining
F-actin was visualized using rhodamine phalloidin (Molecular Probes,
Eugene, OR) as previously described (Xu
et al., 2000).
Apoptosis assay
Apoptosis was analysed via in situ labeling of DNA fragments using an
ApopTag Plus Fluorescein kit (Intergen, Purchase, NY) according to the
manufacturer's instructions for cryosections. A negative control slide was
prepared by omitting the deoxynucleotidyl transferase in the reaction mix. A
positive control was prepared by pretreating with DNase I (Roche Molecular
Biochemicals, Indianapolis, IN) for 5 minutes at room temperature prior to the
addition of the reaction mix.
BrdU labeling
24 hours prior to sacrifice, wild-type and heterozygous SeyDey mice were
given an intraperitoneal injection of BrdU (10 mM stock solution used at 1 ml
per 100 g body weight). Fresh, frozen eye sections were prepared, fixed and
washed in PBS as described above. The sections (10 µm) were incubated in 2
N HCl in 0.5% Triton X-100 for 30 minutes, followed by a second incubation of
fresh 2 N HCl in 0.5% Triton X-100 containing 1 mg ml1
pepsin (Sigma P7012, St Louis, MO) for 10 minutes. The slides were washed with
PBS three times for 10 minutes each, blocked in 10% normal rabbit serum in a
humidified chamber and then incubated with a rat monoclonal anti-BrdU antibody
(Ab) (1:100; Accurate Chemical and Scientific, Westbury, NY) overnight at
4°C. Complete processing of the sections was achieved using a rat
Vectastain Elite ABC kit and diaminobenzidine as described previously
(Davis and Reed, 1996).
Western blot analysis
Mice were euthanized and whole eyes were enucleated and placed in chilled
PBS on ice. Using a dissection microscope, corneas were isolated from the rest
of the eye by introducing a small opening at the limbus with fine forceps and
then separating the cornea from the conjunctiva by pulling on either side of
the opening with fine forceps. The corneas were trimmed free of any remaining
non-corneal tissues with a scalpel blade, frozen on dry ice and stored at
80°C until further use. Tissues were solubilized in 150 mM NaCl, 50
mM Tris, pH 7.4, 0.5% NP-40, 0.5% sodium deoxycholate, 5 mM EDTA, 0.25% SDS,
pepstatin, leupeptin, PMSF and aprotinin. Protein concentration was determined
using the Bio-Rad Protein assay (Bio-Rad, Hercules, CA). PAGE was performed
using precast gels, buffers and 2x Tris-glycine SDS sample buffer
containing 50 mM dithiothreitol followed by transfer to PVDF membrane in
Tris-glycine transfer buffer according to the manufacturer's recommendation
(Novex: Invitrogen, Carlsbad, CA). The membrane was stained with Ponceau Red
(where applicable), blocked in 5% nonfat dry milk in Tris-buffered saline
(Blotto) for 1 hour at room temperature and incubated with primary Ab in
Blotto at 4°C overnight. The membrane was washed with 1x TBS; 0.1%
Tween 20, three times for 15 minutes each. The blot was incubated with a
horseradish peroxidase-conjugated, secondary Ab (1:10,000 in Blotto, Amersham
Pharmacia Biotech, Piscataway, NJ) for 30 minutes. After washing, the
immunoreactive complex was visualized using ECL Plus (Amersham Pharmacia
Biotech, Piscataway, NJ). The following primary antibodies were used:
anti-desmoglein, anti-paxillin, anti-ß-catenin and anti--catenin
(1:1000, 1:10,000, 1:2000, 1:500, respectively; D28120, P13520, C26220,
C19220, Transduction Laboratories, Lexington, KY), anti-E-cadherin (2 µg
ml1; 13-1900, Zymed Laboratories, San Francisco, CA) and
anti-tubulin (1:500; T3526, Sigma, St Louis, MO).
In situ hybridization
Fresh, frozen, 10 µm eye sections were fixed in 4% paraformaldehyde,
treated with proteinase K (0.2 µg ml1 PBS) for 8-10
minutes and processed for in situ hybridization as described previously
(Borchelt et al., 1996).
Riboprobes were synthesized using a DIG RNA Labeling Kit (Sp6/T7) (Roche
Molecular Biochemicals, Indianapolis, IN) with linearized,
proteinase-K-treated, full-length plasmid cDNA templates for mouse K12.
Hybridizations were carried out at 55°C using 200 ng sense or antisense
riboprobe per ml hybridization buffer. Detection of digoxigenin-labeled
hybridization was achieved using an alkaline phosphatase (AP)-conjugated
anti-digoxigenin Ab at a 1:500 dilution followed by the addition of nitroblue
tetrazolium salt and bromo-4-chloro-3-indolylphosphate toluidinium salt. The
reaction was allowed to proceed until a purple color was visible (
2
hours), at which time reactions for both the sense and antisense riboprobes
were terminated.
Cornea fragility assay
The fragility of the corneas was assessed as described previously
(Kao et al., 1996). Briefly,
mice were euthanized and the cornea of the right eye was immediately brushed
gently three times with a PBS-saturated microsponge (K20-5010, Katena
Products, Denville, NJ). The left eye was not brushed. Several drops of
fluorescein (2% in PBS; F-6377, Sigma, St Louis, MO) were applied to both eyes
for 1 minute and then the eyes were washed repeatedly with PBS. The eyes were
examined within 3 minutes to avoid diffusion of the fluorescein and
photographed using a Zeiss Stemi SV11 fitted a Spot camera with a GFP filter
(485/20 excitation).
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Results |
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Postnatal and adult SEY (+/) mouse corneas are abnormal
Histological analysis of adult eye sections revealed that corneas from SEY
(+/) mice were defective when compared with wild-type siblings. The
corneal epithelium was eight to ten cell layers thick in adult wild-type mice
(Fig. 2A), independent of the
strain, compared with 1-7 cell layers in the SEY (+/) mouse. Extreme
reductions in epithelial thickness are shown for adult
Pax6SeyDey (Fig.
2B) and Pax6SeyNeu
(Fig. 2C) mice. The reduction
in corneal epithelial thickness occurred across the entire corneal surface and
varied from mouse to mouse, even between eyes from the same animal, but was
always reduced relative to wild-type corneas. Additionally, the characteristic
morphology of individual cell layers of the corneal epithelium was altered in
the SEY (+/) epithelium. In general, the basal cells were more rounded
and the cell layers were not packed as tightly as the wild-type
counterparts.
|
We confirmed that cells in the corneal epithelial region were bona fide
corneal epithelial cells based on their expression of K12 mRNA and protein, a
marker for corneal but not conjunctival epithelial cells
(Liu et al., 1994;
Moyer et al., 1996
). A robust
hybridization signal was observed for K12 mRNA in wild-type corneal
epithelium using an antisense (Fig.
3A) but not a sense (Fig.
3B) riboprobe. A variable K12 hybridization signal, often
reduced, was observed in most SEY (+/) corneal epithelium
(Fig. 3E,H). Similarly, an
immunoreactive signal of variable intensity relative to the wild type
(Fig. 3C) was also observed in
the corneal epithelium of SEY (+/) mice
(Fig. 3F,G,I,J) using two
different antibodies raised against K12. Specific reactivity of the N-terminal
K12 antibody with corneal but not conjunctival epithelium is shown in
Fig. 3K. Immunoreactivity of
anti-K12 antibody with stromal fibroblasts is nonspecific (W. Kao, personal
communication) and disappears at lower dilutions of antibody (data not shown).
Thus, the expression of K12 mRNA and protein using three independent
means of detection indicate that these cells retain features that are
characteristic of corneal epithelial cells. K12 staining was not detected in
corneas with reductions in corneal epithelial thickness to one or two cell
layers; instead, these cell layers were positive for keratin 4 (K4), a
conjunctival epithelium-specific keratin
(Kurpakus et al., 1994
) (data
not shown). Thicker epithelia were negative for K4 staining (data not shown).
No goblet cells were observed in the two strains of SEY mice used in this
study.
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Other regions of the SEY (+/) cornea were abnormal. Immunostaining
using anti-laminin antibodies revealed that the basement membrane was intact.
However, large aggregates of laminin also appeared in the anterior region of
the SEY (+/), but not the wild type, corneal stroma
(Fig. 4). In 20% of the
SEY (+/) corneas, the stroma showed deviations in thickness and a
general disorganization (Fig.
2). Like the wild-type corneal endothelium, the SEY (+/)
corneal endothelium was intact as a monolayer as evaluated by both light
(Fig. 2) and electron (data not
shown) microscopy.
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One feature of squamous epithelial cell stratification is the establishment of cell layers in an orderly fashion. A time course of stratification in the wild-type C57BL/6 mouse showed that the corneal epithelium is two cell layers thick at birth and develops postnatally to its adult size (6 weeks) of eight to ten cell layers as revealed by hematoxylin and eosin staining (data not shown). Between PN14 and PN21, the epithelium expands by four to six cell layers, however, neither the PN14 nor the PN21 SEY (+/) corneal epithelium was as stratified as a same-age, wild type epithelium (data not shown). These results are consistent with our findings in the adult SEY (+/) cornea and suggest that the process of establishing or maintaining the corneal epithelium with an age-appropriate number of cell layers is impaired as early as the second week after birth in the SEY (+/) mouse.
Changes in proliferative or apoptotic rates do not account for
reduced corneal epithelium thickness in the SEY (+/) mouse
Several avenues of investigation were taken to ascertain whether the SEY
corneal epithelium phenotype results from changes in the growth status of the
epithelial cells. First, we determined whether a reduction in cell
proliferation, as measured by DNA synthesis, could account for the abnormal
corneal phenotype. Mice were harvested 24 hours following an intraperitoneal
injection of BrdU and the eyes were processed for immunohistochemistry using
an anti-BrdU antibody. The number of BrdU-labeled cells was divided by the
total number of epithelial cells to determine the proliferative index (PI).
Surprisingly, the PI of the Pax6SeyDey (+/)
epithelium was greater than that of wild-type siblings at 6 weeks of age
(Fig. 5). A PI of 45% in
Pax6SeyDey (+/) compared with 4% in wild-type
epithelium indicated that more cells have entered S phase in the mutant
(+/) epithelium. Similar PIs were calculated for PN21
Pax6SeyDey (+/) and wild-type corneas (data not
shown).
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The possibility that a reduction in the layers of the cornea was due to an increase in cell turnover caused by apoptosis was analysed by TUNEL labeling of the corneal epithelium in PN21 and adult mice. There was no difference in the number of TUNEL-positive cells between wild-type and SEY (+/) corneas at either age; only zero to two labeled cells were observed per section, in contrast to the positive control slide, in which all nuclei were labeled following pretreatment with DNase (data not shown).
The roles of EGFR and ErbB2, two growth-factor receptors previously shown
to mediate corneal epithelial cell proliferation
(Savage and Cohen, 1973;
Xie et al., 1999
), were
examined by immunohistochemistry. The pattern of immunostaining for ErbB2
appeared similar in the SEY (+/)
(Fig. 6B) and wild-type
(Fig. 6A) corneal epithelial
cells. However, the staining pattern for EGFR differed between SEY (+/)
(Fig. 6D) and wild-type
(Fig. 6C) corneas: large
aggregates of immunoreactive product for EGFR were observed in the nuclei of
the SEY (+/) but not the wild-type cells at 6 weeks of age. Expression
analysis of EGFR and ErbB2 by semiquantitative reverse-transcription PCR,
showed no significant difference between the levels of mRNA expression in the
SEY (+/) and wild-type corneas (data not shown).
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Defects in cellular adhesion contribute to reduced thickness of the
SEY (+/) cornea epithelium
Electron microscopy revealed that the close adhesion of the cell layers
normally present in the corneal epithelium was absent in the SEY (+/)
epithelium (Fig. 7). Large gaps
appeared between cells that remain attached to each other through larger than
normal desmosomal complexes (Fig.
7B,D). These abnormalities were restricted to the suprabasal,
middle and superficial layers of the epithelium (data not shown). The SEY
(+/) corneal cells also appeared swollen.
|
To investigate the nature of the defect in adhesion, we examined the levels
and/or locations of several molecules that are known to be important in the
two major classes of anchoring junctions. Western blot analysis showed
equivalent levels of E-cadherin (the transmembrane core of epithelial adherens
junctions) in total corneal extracts from wild-type and SEY (+/) mice
(Fig. 8A). Moreover, there was
no difference between the plasma membrane locations of E-cadherin in wild-type
(Fig. 8G) and SEY (+/)
(Fig. 8H) corneal epithelial
cells by immunofluorescence. By contrast, the level of desmoglein, the major
cadherin found in desmosomes, was reduced eight times in SEY (+/)
relative to wild-type corneas (Fig.
8B). The amounts of ß- and -catenin, additional
components of anchoring junctions, were also reduced, about two times, in
mutant SEY (+/) cornea (Fig.
8C,D, respectively). Equal loading of soluble corneal extracts was
confirmed by staining duplicate lanes on the blot with Ponceau Red
(Fig. 8F). Most protein bands
show equivalent intensity except for the 54 kDa band (corresponding to
aldehyde dehydrogenase class 3, an abundant corneal epithelial protein), which
is also reduced in the SEY (+/) cornea (J. Davis and J. Piatigorsky,
unpublished). Abundant, equivalent amounts of transketolase at 68 kDa indicate
that the extracts were derived primarily from corneal rather than conjunctival
epithelium, which has low basal levels of transketolase
(Guo et al., 1997
).
|
Although there were no visible gaps in the basal and suprabasal cell
layers, the presence of molecules mediating adherence to the extracellular
matrix via hemidesmosomes and focal adhesions was examined. Localization of
the 5,
6, ß1 and ß4 integrin subunits using specific
antibodies revealed similar, basement membrane staining in the corneas of
wild-type and SEY (+/) mice (data not shown). Western blot analysis
showed that the amount of paxillin, a protein associated with focal adhesions,
was similar in SEY (+/+) and SEY (+/) corneas, except for a small
upwards shift in electrophoretic migration in paxillin from the SEY
(+/) cornea (Fig. 8E), a
possible post-translational modification.
Wild-type and SEY (+/) corneas fluoresced with a similar pattern after staining with rhodamine-conjugated phalloidin, showing that the actin cytoskeleton was intact (Fig. 9A,B, respectively). By contrast, the intermediate filament K12 mRNA and protein was reduced in the corneal epithelium in >90% of SEY (+/) compared with wild-type mice (Fig. 3). These results, taken together, suggest that desmosomes and the intermediate filaments to which they attach are abnormal and contribute to a loss of adhesion in the SEY (+/) corneal epithelium.
|
Finally, we tested whether the epithelial cells of SEY (+/) corneas adhere more loosely than those of the wild-type corneas. Gentle rubbing with a microsponge saturated with PBS removed the corneal epithelium of SEY (+/) mice but equivalent rubbing did not affect the corneal surface of wild-type mice (Fig. 10). The bright staining in the SEY (+/) relative to the wild-type cornea represents an epithelial defect. Fluorescein staining occurs when there is a loss of cells at the epithelial surface. Epithelial cell loss in SEY (+/) eyes after rubbing was confirmed by light microscopy (data not shown). Even without rubbing, fluorescein staining often revealed diffuse, punctate defects in the corneas of SEY (+/) mice (Fig. 10C).
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Discussion |
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Our data show that cell-cell junctions, known to be important and plentiful
in epithelial cells (Albert and Jakobiec,
1994), are altered in SEY (+/) corneal epithelia.
Desmosomes in the SEY (+/) cornea formed larger complexes than their
wild-type counterparts. Coincident with changes in the desmosomal
ultrastructure was the widening of intercellular spaces between cells. These
defects were most apparent in the superficial, middle and suprabasal layers of
the SEY (+/) corneal epithelium, consistent with the finding that
desmosomes are denser in the superficial layers of the corneal epithelium
(Hogan et al., 1971
). A
similar phenotype was observed in the skin of transgenic mice overexpressing a
mutant form of Dsg3 (Allen et al.,
1996
). Our results are also compatible with clinical symptoms in
patients with phemphigus vulgaris, who suffer from losses in cell adhesion in
squamous epithelia including ocular tissue as a result of raising
autoantibodies against Dsg3 (Allen et al.,
1996
; Koch et al.,
1997
; Smolin and Thoft,
1994
).
Concomitant with the ultrastructural changes in the desmosomes, we also
found that the level of Dsg, an adhesion molecule that forms the transmembrane
core of desmosomes, was reduced in the SEY (+/) cornea (Angst, 2001).
The amounts of individual desmosomal proteins appears to be crucial, because
desmocolin1 (Chidgey et al.,
2001) or Dsg 3 (Koch et al.,
1997
) null mice form `normal-looking' desmosomes but their skin
eventually loses adhesion and falls off.
In contrast to desmosomal cadherins, the levels of E-cadherin (the
transmembrane core of adherens junctions) and its localization to the plasma
membrane were similar in wild-type and mutant SEY corneas. However, two other
structural components of adherens junctions, ß- and -catenin, were
reduced in SEY (+/) corneal cells.
-Catenin has been shown to
increase adhesion in vitro (Marcozzi et
al., 1998
) and in vivo
(Bierkamp et al., 1999
). It is
possible, therefore, that the decrease in catenins in SEY (+/) cornea
further reduces the ability of the remaining junctions to provide strong
intercellular adhesion.
A decrease in K12, the major intermediate filament-forming keratin of mouse
corneal epithelial cells (Liu et al.,
1993; Liu et al.,
1994
), might also contribute to loss of adhesion. The anchoring of
the intermediate filaments to the desmosomes produces supracellular
scaffolding that is essential in maintaining epithelial tissue integrity
(Kivela and Uusitalo, 1998
;
Kowalczyk et al., 1999
;
Smith and Fuchs, 1998
;
Steinberg and McNutt, 1999
;
Troyanovsky and Leube, 1998
;
Vasioukhin and Fuchs, 2001
).
Gentle rubbing of the corneal epithelium produced mild erosion of the SEY
mutant similar to that observed in K12 homozygous-null mice
(Kao et al., 1996
).
A role for Pax6 in cell adhesion has been suggested by previous studies
(Brunjes et al., 1998;
Chapouton et al., 1999
;
Collinson et al., 2000
;
Dohrmann et al., 2000
;
Estivill-Torrus et al., 2001
;
Gotz et al., 1996
;
Grindley et al., 1997
;
Mastick et al., 1997
;
Matsuo et al., 1993
;
Quinn et al., 1996
;
St-Onge et al., 1997
;
Stoykova et al., 1996
;
Stoykova et al., 1997
;
Warren et al., 1999
).
Transcriptional targets of Pax6 include genes controlling cell-cell
interactions (Chalepakis et al.,
1994
; Duncan et al.,
2000
; Edelman and Jones,
1995
; Holst et al.,
1997
; Meech et al.,
1999
). We propose that Pax6 has a role in normal turnover of the
corneal epithelium by maintaining, directly or indirectly, various factors
that contribute to adhesion. In connection with this, we find that Pax6 is not
detected in the outermost cell layer of the corneal epithelium
(Fig. 1), coincident with the
appearance of gaps between this layer and the layer below it
(Fig. 7). Based on the current
study, a decrease in Pax6 would lead to a reduction in adhesion conducive to
the natural sloughing of the most superficial layer of the cornea.
The most plausible explanation for an increased proliferative index in the
Pax6SeyDey (+/) corneal epithelium is that the
barrier has been perturbed because of the loss of cells from the upper layers,
a condition known to induce cell proliferation
(Chung et al., 1999;
Cotsarelis et al., 1989
;
Zieske, 2000
). Further, the
EGFR is found localized as large aggregates in the cytoplasm and nucleus
following corneal wounding (Zieske et
al., 2000
), as was observed in the SEY (+/) corneal
epithelial cells. Because the Pax6 gene and flanking chromosomal
regions are deleted in the Pax6SeyDey strain, it is also
possible that the proliferative changes noted here are due to genes other than
Pax6.
The persistence of K12 gene expression in most SEY corneas in our
investigation indicates that the SEY (+/) epithelial cells are
authentic corneal epithelial cells. Nevertheless, a few corneas produced K4 in
the corneal epithelial region of the ocular surface, but only if there was a
severe reduction in the epithelium (to one or two cell layers), suggesting
that conjunctivalization, a response associated with a deficiency in limbal
stem cells and known to occur in human aniridia patients, might also occur in
the small-eye syndrome of the mouse
(Daniels et al., 2001;
Dua and Azuara-Blanco, 2000
;
Moyer et al., 1996
). In the
present study, several reduced-thickness epithelia were immunoreactive for K4
on the top and K12 on the bottom cell layer (data not shown), suggesting that
a dynamic process of corneal epithelial loss and conjunctival epithelium
resurfacing might take place in some cases. These results warrant further
investigation into a role for Pax6 in limbal stem-cell number and the corneal
infiltration of conjunctival cells in the SEY mouse model.
Eye development depends on the coordinated interaction of many tissues. The
inductive action of lens on corneal development obscures an understanding of
whether Pax6 has a primary effect in the cornea, independent of its effect in
the lens (Beebe and Coats,
2000; Genis-Galvez,
1966
; Genis-Galvez et al.,
1967
; Grainger,
1992
; Kidson et al.,
1999
; Reneker et al.,
2000
; Zinn,
1970
). Future experiments analysing mice overexpressing
Pax6 specifically in the corneal epithelium beginning at eye opening
will hopefully provide an answer to whether Pax6 has a direct, primary role in
the generation and maintenance of the adult, stratified corneal
epithelium.
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Acknowledgments |
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