First published online 8 April 2003
doi: 10.1242/jcs.00441
Requirement for Pax6 in corneal morphogenesis: a role in adhesion
Janine Davis1,
Melinda K. Duncan2,
W. Gerald Robison, Jr3 and
Joram Piatigorsky1,*
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
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Fig. 1. Pax6 expression in C57Bl/6 mouse cornea. Localization of Pax6
protein to the corneal epithelium, but not the stroma or endothelium, is shown
by bright-field microscopy using an antibody against Pax6 on cryosections from
the central (A) and peripheral (B) corneal regions of a 6-week-old wild-type
mouse. No primary antibody used (C) serves as a control. Higher magnification
of central cornea (D) showed nuclear localization of Pax6, indicated by the
brown immunoreactive product. DAPI counterstaining of nuclei (E) (on the same
section as in D) revealed that most corneal epithelial cells produce Pax6,
indicated by the quenching of DAPI fluorescence by the brown Pax6-dependent
immunoreactive product. Epithelial cells in the most anterior layer of the
cornea, however, fluoresce brightly (asterisks), indicating that Pax6 is
reduced in this cell layer. Abbreviations: c, corneal epithelium; k,
keratocyte; n, endothelium; s, stroma.
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Fig. 2. Morphology of corneas from adult SEY mice. Corneas were visualized by
bright-field microscopy using hematoxylin and eosin. Same-magnification views
of corneas from wild-type (A), Pax6SeyDey (+/–) (B)
and Pax6SeyNeu (+/–) (C) adult (6 weeks) mice showed
severe reductions in corneal epithelial thickness in the SEY (+/–) mice.
Abbreviations: c, corneal epithelium; n, endothelium; s, stroma.
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Fig. 3. Keratin 12 (K12) in corneas from wild-type and SEY (+/–) mice.
K12 mRNA was detected by in situ hybridization in the corneal
epithelium of wild-type (A,K) and SEY (+/–) (E,H) mice using an
antisense riboprobe. A control using a sense probe is shown for a wild-type
mouse (B). K12 protein was detected by immunostaining with an anti-N-terminal
K12 antibody in wild-type (C) and SEY (+/–) (F,I) corneal epithelium and
with an anti-C-terminal K12 antibody in SEY (+/–) (G,J). No primary
antibody used (D) serves as a control. The specificity of the N-terminal K12
antibody for corneal but not conjunctival epithelium is shown in K.
Abbreviations: c, corneal epithelium; cn, conjunctival epithelium.
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Fig. 4. Localization of laminin in SEY (+/–) mouse corneas. Laminin was
detected by immunofluorescence in the basement membrane underlying the corneal
epithelium and the endothelium of wild-type (A) and mutant (+/–)
Pax6SeyDey (B) corneas. The dim fluorescence present in
wild-type epithelial cells was absent in mutant cells, and large aggregates of
immunofluorescence (asterisks in B) occasionally appeared in the stroma of
mutant corneas. Similar staining was observed in the
Pax6SeyNeu (+/–) cornea (data not shown).
Abbreviations: c, epithelium; s, stroma; n, endothelium.
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Fig. 5. DNA synthesis in wild-type and Pax6SeyDey (+/–)
mouse corneas. Mice were sacrificed 24 hours after an injection of BrdU and
corneas were processed for immunohistochemistry using an anti-BrdU antibody.
Approximately ten times more BrdU-positive cells (black signal indicated by
arrows) are detected in 6-week-old SEY (+/–) mouse corneal epithelium
(B) than in wild type (A) corneal epithelium. BrdU-positive cells are
generally located on the basal side of the wild-type epithelium, whereas they
are distributed uniformly in the SEY (+/–) epithelium. The brown color
is observed in a control section of PN21 wild-type cornea (C), where no
anti-BrdU antibody was used and is considered to be background. Abbreviations:
c, epithelium; s, stroma.
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Fig. 6. Corneal epithelial localization of ErbB2 and epidermal-growth-factor
receptor (EGFR). Wild-type (A,C) and SEY (+/–) (B,D) corneas of
6-week-old mice were incubated with antibodies against ErbB2 (A,B) or EGFR
(C,D). Immunostaining patterns for ErbB2 were similar in the wild-type and
mutant cornea. However, there is a change in EGFR localization as revealed by
a strong, diffuse staining pattern in the wild type versus the pronounced
aggregation of immunoreactive product in the nuclei of SEY cells. As a
control, a section of wild-type adult cornea (E) was processed exactly as
above except that no primary antibody was used. Abbreviations: c, epithelium;
s, stroma.
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Fig. 7. Ultrastructure of wild-type and SEY (+/–) corneal epithelium.
6-week-old wild-type (A,C) and SEY (+/–) (B,D) corneal epithelium are
shown at 9900x (A,B) and 15,000x (C,D) magnification. Large gaps
(asterisks in B,D) were present between cells from the suprabasal to
superficial layers in SEY (+/–) epithelium. By contrast, notice the
tightly adhered cell layers in the wild-type epithelium; gaps (asterisk in C)
are present only in the most superficial layer of the wild-type epithelium.
Furthermore, the number of individual desmosomal complexes (the electron-dense
structures; arrows in C,D), are reduced, but the size of these complexes is
increased in the SEY (+/–) compared with the wild-type epithelium.
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Fig. 9. Cytoskeletal marker in wild-type and SEY corneas. The actin cytoskeleton
was similar in wild-type (A) and SEY (+/–) (B) corneal epithelium of
6-week-old mice as revealed by rhodamine-phalloidin. Abbreviations: c,
epithelium; s, stroma.
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Fig. 10. Corneal epithelial defects after brushing the corneal surface with a
microsponge. Wild-type (A,B) and SEY (+/–) (C,D) eyes from 6-week-old
mice were untreated (A,C) or treated (B,D) by brushing the corneal surface
with a microsponge. The eyes were then stained with fluorescein, washed and
photographed. After brushing, intense, diffuse fluorescein staining was
observed in the SEY (+/–) cornea (D), in contrast to that observed in
the wild-type cornea (B). Abbreviation: c, cornea.
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© The Company of Biologists Ltd 2003
-catenin (D) and paxillin (E) derived from wild-type and SEY
(+/–) corneal extracts from 6-week-old mice. Levels of desmoglein and
ß- and