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The Pax-6 Homeobox Gene Is Expressed Throughout the Corneal and Conjunctival Epithelia Barba M. Koroma* Jun-Ming Yang* and OlofH. Sundin*-\ Purpose. Heterozygous defects in the highly conserved PAX6 homeobox gene are associated with aniridia, an inherited human disorder affecting several ocular structures, including the adult cornea. This work establishes the pattern of Pax-6 gene expression in the surface epithelia of the late embryonic and adult eye. Methods. Chick embryo sections and wholemounts, as well as adult mouse and monkey tissues, were analyzed by in situ hybridization and immunohistochemistry with probes specific to Pax6. Western immunoblots were used to detect Pax-6 protein, and mRNA expression was analyzed by quantitative reverse transcription-polymerase chain reaction. Remits. In days 5 and 6 chick embryos, Pax-6 protein is found in the nuclei of all cells within the corneal epithelium and in the future conjunctiva. Although not detected in the cornea by in situ hybridization, Pax-6 mRNA is, in fact, present at levels comparable to those observed in the retina. In the mature mouse, Pax-6 protein was expressed in all cells of the corneal epithelium, the limbus, and the entire conjunctiva. Similar results were obtained for the monkey cornea. Conclusions. These data indicate that in additiontoits role in the embryo, Pax-6 is expressed strongly in surface epithelia of the adult cornea and conjunctiva. In cells of these tissues, the gene may function by regulating structural or secretory specializations. Pax-6 might play a direct role in the maintenance and proliferation of corneal stem cells, a vital process that appears to be defective in aniridia. Invest Ophthalmol Vis Sci. 1997;38:108-120. From the * Wilmer Eye Institute and the \Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland. Supported by a grant from the Knights Templar Eye Foundation; a grant from Fight For Sight (Research Division of Prevent Blindness America) in memory of Mary E. and Alexander P. Hindi; and National Institutes of Health grant R01 EY10729 (ONS). BMK is supported by an NIH Minority Postdoctoral Training Supplement. OHS is the recipient of a Research to Prevent Blindness Career Development Award. Received for publication April 5, 1996; revised August 6, 1996; accepted August 28, 1996. Proprietary interest category: N. Reprint requests: OlofH. Sundin, Wilmer Eye Institute, The Johns Hopkins Institution, 840 Maumenee Building, 600 N. Wolfe Street, Baltimore, MD 21287. and mouse8 and later was identified as the site of mutations responsible for human aniridia9'10 and the phenotypically similar small eye (Sey) mutant of the mouse.1112 Individuals heterozygous for mutations in human PAX6 often develop eyes with a greatly reduced iris and with abnormalities of the retina, lens, and cornea.91013 Those rare individuals homozygous for mutations in PAX6 are born without eyes and die shordy afterward, demonstrating that diis gene is essential to human ocular development.14 In mice12 and rats,15 viable heterozygous animals show ocular abnormalities, including reduction of the iris, whereas homozygotes are anophthalmic, lack nasal epithelia, and die shordy after birth. Interestingly, die earliest visible defect in embryos homozygous for mutations in Pax6 is the absence of a lens placode.11'16 This is consistent with the observation that lens-competent regions within the future head ectoderm of the neural-plate stage embryo are the first sites at which chicken and mouse Pax-6 are expressed during development and that the early lens placode strongly expresses Paxg i6,i7 -pjie f a j m r e o f e y e formation in Pax-6 homozygous mutant mice appears direcdy linked to absence 108 Investigative Ophthalmology & Visual Science, January 1997, Vol. 38, No. 1 Copyright © Association for Research in Vision and Ophthalmology 1 he vertebrate eye develops through a complex series of events, beginning with early embryonic primordia located in the head ectoderm and central nervous system. ' Insight into the molecular basis of this process has been provided recendy by the study of homeobox genes,3'4 a large family of structurally related genes that are among the key determinants of cell fate and body plan in vertebrates and invertebrates.0'6 The homeobox genes encode transcription factors and function by regulating the expression of other genes. Pax-6 is a homeobox gene known to play an especially important role in several aspects of eye development. This gene was cloned originally in zebrafish7 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933195/ on 05/05/2017 Pax-6 in Ocular Surface Epithelia 109 METHODS of the early lens placode, which plays an essential role in organizing the optic cup.1'2 During later developIsolation, Fixation of Embryos, and Ocular ment, Pax-6 continues to be expressed in the proliferTissues ating anterior lens epithelium. It also is expressed in the undifferentiated retina and in the differentiating Day 4 (HH stage 18see 28 ), day 5, and day 6 chick (Gallus amacrine and ganglion cells, suggesting that it has a gallus) embryos were fixed overnight in freshly chilled role in ocular tissues of neural as well as ectodermal MEMFA (0.1 M MOPS, pH 7.4, 2 mM EGTA, 1 mM origin.8 MgSO4, 3.7% formaldehyde) ( see29 ). The tissue was The protein encoded by Pax-6 contains a homeorinsed in 0.9% NaCl, equilibrated in 90% methanoldomain and a paired domain, two DNA-binding motifs water, and stored at — 20°C. Eyes of embryonic days required for directing the transcription factor to its 10, 13, and 15 chick were dissected from the head, target sites in the genome.89'18'19 The structure and opened, and fixed. Freshly killed 30-day-old mice were function of these domains has been extremely conprovided by the laboratory of Dr. Ruben Adler of the served through vertebrate evolution, with near idenWilmer Eye Institute (Baltimore, MD). Each mouse tity between the human and fish.710'18 These domains was chilled immediately on ice and the entire orbit, are highly conserved in eyeless, a homologous Drosoph- attached muscles, and eyelids were dissected from the ila gene essential for eye development.4 This Drosophila head and placed in ice-cold MEMFA for overnight gene has the remarkable ability to direct other regions fixation at 4°C. Tissues were from a young adult male cynomolgus monkey (weight, 11.5 pounds) killed as of the insect body to develop complete ommatidial part of a glaucoma study conducted by Dr. Harry Quigeye structures.20 Concerning its regulatory functions, ley of the Wilmer Eye Institute. The anterior segment there is evidence that the vertebrate Pax-6 gene binds of the control eye, which normally would not have and activates its own promoter.21 Pax-6 is known to been used in the glaucoma experiments, was placed regulate directly the transcription of lens crystallin immediately into ice-cold MEMFA fixative overnight, genes, including zeta (guinea pig),22 alpha A then stored in 90% methanol at -20°C as before. Tis(mouse),23 and delta crystallin (chicken).24 As noted sues were fixed and stored in the same manner for earlier, patterns of transcription17 and genetic eviboth in situ hybridization and immunohistochemistry. dence" 16 implicate Pax-6 in several aspects of lens The care of experimental animals was in accordance development or regeneration.25 Because Pax-6 expreswith ARVO Statement for the Use of Animals in Ophsion is first required for lens placode development at thalmic and Vision Research. a stage well before the activation of crystallin 1116 genes, Pax-6 must regulate additional genes required for the formation of the lens. In Situ Hybridization The lens is not the only ectodermal structure of In situ hybridization with digoxigenin-labeled Pax-6 the eye that depends on the Pax-6 gene for its normal riboprobe was carried out as described earlier.17 The development. In the early optic vesicle-stage chick em512-bp probe was derived from a chicken Pax-6 cDNA bryo, Pax-6 is expressed strongly in two bilateral and encoded the 170 carboxy-terminal amino acids of patches that include the future lens but that extend the protein.17 Stage 18 (early day 4) and day 5 chick 17 beyond its boundaries. As the center of the lens placembryos were dissected in ice-cold phosphate-bufode invaginates to form the lens vesicle, the peripheral fered saline and fixed overnight in MEMFA at 4°C. ectoderm remaining on the surface shows substantial Stage 18 embryos were processed for wholemount in expression of Pax-6 mRNA. This peripheral ectoderm situ hybridization, and this was followed by embedding of the lens placode is fated to contribute to the corneal in JB-4 glycolmethacrylate resin (Polysciences, Warand conjunctival ectoderm.1'2 Given the fact that some rington, PA) and sectioning at 10 (im.17 Day 5 and individuals with aniridia develop serious abnormalities later embryos were equilibrated in a solution of 20% of the cornea in adulthood, we were interested in sucrose and 20 mM Tris, pH 7.4; this was followed by determining whether in animal models, Pax-6 contintransfer to OCT medium (Miles Laboratories, Elkhart, ues to be expressed in these anterior tissues. One of IN) and by freezing and cryo-sectioning at — 25°C. In situ hybridization of sections was carried out with the these abnormalities is the corneal pannus, a localized same hybridization and wash times used for the opacification of the cornea often accompanied by an wholemount technique. For in situ hybridization of ingrowth of vascular tissue.13'26 Other problems associwholemounts and sections, all hybridization and strinated with aniridia include cataracts and glaucoma.27 gency washes were performed at 63°C in 50% forIn this study, we report that Pax-6 protein and mamide containing solutions (see refs. 17, 29 for demRNA are expressed at high levels in the adult corneal tails). Alkaline phosphatase color reaction was perepithelium. We also show that this domain of expresformed for 16 hours. Sections were postfixed in sion includes the conjunctiva, thus marking the entire MEMFA for 30 minutes at room temperature, then compartment of the ocular surface. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933195/ on 05/05/2017 110 Investigative Ophthalmology & Visual Science, January 1997, Vol. 38, No. 1 rinsed and stored overnight in methanol (90%) at -20°C. Immunohistochemical Analysis Antibodies specific for Pax-6 were generously provided by Drs. Janine Davis and Randall Reed of the Howard Hughes Medical Institute at Johns Hopkins University School of Medicine (Baltimore, MD). The antiserum was prepared by immunizing rabbits with a bovine serum albumin-conjugated synthetic peptide corresponding to the 17 carboxy terminal amino acids of mouse Pax-6. The chick C-terminal sequence is identical to that of mouse.8'30 The antiserum was affinity purified by binding to immobilized peptide. Tissue fixation and cryosectioning were as described for in situ hybridization. Six-day chick embryo heads were fixed, as described, then embedded in 2% low melting temperature agarose-phosphate-buffered saline and sliced into 100 ^m sections using a vibratome. Immunohistochemistry was performed in a basic mixture of 5% nonfat milk dissolved in TST (10 mM Tris, 150 mM NaCl, 0.1% Tween 20) and was clarified by centrifugation at 14,000 RPM for 10 minutes, essentially as described earlier.31 Sections were first blocked in 5% milk-TST for 2 hours at room temperature, then incubated overnight at 4°C in the same solution containing a 1:400 dilution of Pax-6 antibody. Sections were washed three times in TST (without milk) for 10 minutes each wash and incubated in a 1:200 dilution of biotinylated goat antirabbit IgG (Vector Laboratories, Burlingame, CA) for 3 hours at room temperature. Sections were washed three times in TST for 30 minutes each time and incubated in a 1:500 dilution of avidin-horseradish peroxidase-conjugated tertiary antibody solution at 4°C overnight. Peroxidase color reaction was performed using the VIP kit (Vector Laboratories) and photographed using a Zeiss SV-11 or Axioskop microscope (Carl Zeiss, Thornwood, NY). Western Immunoblot Analysis Corneal epithelia were isolated from eyes of day 15 chick embryos by microdissection using electrolytically sharpened tungsten needles. During the dissection, anterior segments were immersed in ice-cold phosphate-buffered saline containing 20 mM EDTA. EDTA treatment allowed the corneal epithelium to be peeled away from the underlying stroma without the use of proteases. Retina and wing skin from day 15 chick embryos, as well as from day 4 embryo limb bud tissues, were dissected in the same solution. Corneal epithelium was solubilized on ice using a 1 ml dounce homogenizer and 1 ml of 2 X protein gel sample buffer (160 mM Tris-HCl, 4% sodium dodecyl sulfate, 30% glycerol, 5%-mercaptoethanol, 10 mM dithiothreitol, 0.05% bromophenol blue).32 Other tissues were lysed in roughly similar proportions. Lysate was immersed in a boiling water bath for 5 minutes and centrifuged at 14,000 rpm for 10 minutes. Aliquots of protein supernatant were transferred to fresh microfuge tubes and stored at — 80°C until ready for use. Protein quantitation was performed after removal of mercaptoethanol and sodium dodecyl sulfate by trichloroacetic acid precipitation using the BCA (bicinchoninic acid; Pierce, Rockford, IL) technique. Electrophoresis and western immunoblot analyses were performed essentially as described previously.32 Equal concentrations of protein samples were loaded onto a 4% to 20%, 0.75-mm thick precast gradient minigel (Biorad, Melville, NY) and electrophoresed at 2 V/cm. Biotinylated broad-range (6.5 to 200 kDa; Biorad) molecular weight markers were run with the samples. Briefly, proteins were electroblotted onto 0.4-fj.m nitrocellulose membranes (Bioblot, NC; Costar, Cambridge, MA) using a mini-tankblot (Biorad) apparatus for 1 hour at 100 V. Blots were incubated for 2 hours in blocking buffer (5% milk-TST) and transferred to Pax-6 antibody solution diluted 1:200 in blocking buffer and incubated overnight at 4°C. Blots were washed in Tris-buffered saline and incubated for 1 hour at room temperature in a double-secondary antibody solution of antirabbit IgG-horseradish peroxidase and Avidin-horseradish peroxidase (Biorad), diluted in blocking buffer at 1:1000 and 1:2500 ratios, respectively. The blots were washed again in Tris-buffered saline, immersed in ECL reagents as described in Amersham (Arlington Heights, IL) ECL protocols, and exposed to x-ray film. Detection and Quantitation of Pax-6 mRNA by Reverse Transcription -Polymerase Chain Reaction RNA Isolation and cDNA Synthesis. Total RNA was isolated using the Qiagen (Chatsworth, CA) RNeasy miniprep kit. Briefly, tissues isolated as described for immunoblot analysis were homogenized in guanidine isothiocyanate lysis buffer, bound to a column matrix, washed, and eluted in 30 //I water. A basic reverse transcription— polymerase chain reaction protocol33 was modified to obtain relative quantities of Pax-6 mRNA. Reverse transcription was carried out in a 20-//1 mixture containing 2 fi\ of miniprep RNA, 200 U cloned MuLV reverse transcriptase (Gibco BRL), 100 mM KC1, 5 mM MgCl2, 25 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, 1 mM of each dNTP, and 10 /JM of random DNA hexamers (Pharmacia) , and it was incubated at 37°C for 30 minutes. The cDNA mixture was diluted with water to 100 [A, heat denatured at 99°C for 90 seconds, and stored at -20°C. Normalization of Samples to 18s rRNA. Two microliters of each cDNA preparation was added to a standard 20-fA PCR mixture containing 200 mM of each dNTP, Taq polymerase, and 2 /xM each of the oligonucleotide primers TTGGTGACTCTAGATAACCTC and Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933195/ on 05/05/2017 Pax-6 in Ocular Surface Epithelia GGCTCCCTCTCCGGA. These primers were derived from the chicken 18s rRNA sequence under Genbank accession number D38360. Amplification was carried out in the presence of 1 /iCi 32P-dCTP for eight cycles at 94°C for 1 minute, 46°C for 1 minute, and 72°C for 30 seconds. Glycogen carrier was added, and unincorporated nucleotides were removed by precipitation in ammonium acetate.34 The labeled amplimer was mixed with unlabeled carrier amplimer and resolved by agarose gel electrophoresis. Amplimer bands were visualized by ethidium staining, excised, melted in 10 volumes of water at 70°C, and scintillation counted. Based on the relative yield of this amplimer, the cDNA preparations were diluted individually so that they contained the same concentration of 18s rRNA. Quantitation of Pax-6 mRNA. Two microliters of each normalized cDNA solution was added to a 20-fA PCR mixture containing Taq polymerase (Boehringer Mannheim) and 2 (JM each of CCGTGCGACATCTCCCGAATCCTG (sense) and CACGCAAAGATGGAGGGGCACTCTC (ann'sense). These primer sequences correspond to chicken Pax-6 sequences located in the paired box coding region and spanning the exon 4/5 splice junction.30'35 Amplifications were for 16, 25, or 35 cycles at 94°C for 1 minute, 62°C for 30 seconds, and 72°C for 50 seconds. Gels were stained in SYBR Green I (Molecular Probes, Junction City, OR) and photographed under 254 nm ultraviolet light. Protein quantitation, a PCR reaction including 1 ^Ci 32P-dCTP, was amplified for 16 cycles. This reaction did not yield enough amplimer to be visible by SYBR Green staining after electrophoresis and was well within the exponential phase of the PCR reaction.33 The PCR was terminated by the addition of EDTA-containing sample buffer containing unlabeled amplimer as a carrier and marker, then resolved by electrophoresis in 8% acrylamide. Bands were located by staining with ethidium, excised, and scintillation counted. Autoradiography was by exposure to Kodak xray film (Eastman Kodak, Rochester, NY) for 10 days. Relative molar yield of a given cDNA amplimer was determined by subtracting the raw counts (per minute) from a control analysis carried out without cDNA template, and then dividing these net counts by the number of labeled cytosine residues in the amplimer. This relative molar yield of each sample was normalized to the total Pax-6 mRNA detected in retina (set to equal 1). RESULTS Pax-6 mRNA and Protein in the Developing Chick Eye: Immunohistochemistry In the stage 19 chick embryo (early day 4), the lens vesicle has separated fully from the corneal epithelium. At this stage, in situ hybridization demonstrated expression of Pax-6 mRNA in several ocular sites (Fig. 1A). Expression of mRNA at this stage was most promi- 111 nent in the future retina and in the retinal pigment epithelium along the margins of the optic cup. In the lens, signal was detected readily in the anterior lens epithelial cells and in the bow region, but it was absent from the elongating fiber cells. The head ectoderm overlying and surrounding the lens and optic cup also showed significant expression of Pax-6 mRNA. This region of surface ectoderm is fated to generate the corneal ectoderm and conjunctiva. Pax-6 antibody staining of embryos at the same stage revealed a pattern similar to that obtained by in situ hybridization (Fig. IB). As expected for a DNA-binding transcription factor,19'2135 the Pax-6 antibody detected a protein strongly localized to the nucleus. There were, however, differences between the pattern detected with Pax-6 antibody and in situ hybridization probes. Unlike the pattern seen with mRNA, Pax-6 protein was detected readily in cells of the central optic cup and lens fiber layer, although at somewhat lower levels than in regions that prominently express the mRNA. In the head ectoderm, Pax-6 protein was clearly visible in the nuclei of cells in a wider area around the eye (Fig. IB). In the dorsal head ectoderm and in ectoderm outside the head, neither Pax-6 protein or mRNA was detected (not shown). In the anterior segment of a day 5 chick embryo, substantial Pax-6 expression was observed in the anterior epithelium and bow region of the lens (Fig. 1C). Pax-6 mRNA was again absent from the lens fiber layer. When Pax-6 protein was detected using the antibody (Fig. ID), a similar distribution was observed, but the Pax-6-expressing cells tended to extend further into the lens fiber layer, although they were absent from the nuclei of cells in the center of the lens. These differences in the lens and retina might reflect the persistence of Pax-6 protein in the lens fibers and central retina. These are the same cells that expressed the mRNA at significant levels during earlier stages of development.1617'24 Expression of Pax-6 mRNA and protein was found throughout the retinal and pigment epithelial layers of the day 5 optic cup, including the central retina (not shown). In the corneal epithelium and conjunctiva of the day 5 chick embiyo, the nuclei of all cells stained strongly with antibody to Pax-6 protein. It should be noted that Pax-6-specific signal was observed only in the epithelial component of the developing cornea, not in the stroma or endothelium. One striking observation in the examination of the chick embryos between days 4 to 6 is that the domain of Pax-6 expression in the surface ectoderm becomes more sharply defined during development. In early day 4 embryos, the dorsal boundary of the Pax-6 domain appeared as a gradually decreasing gradient, with expressing cells intermixed with nonexpressing cells (Fig. IB). By day 6, however, (Figs. 2A, 2B) this dorsal boundary had become sharply defined and coincided with the edge Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933195/ on 05/05/2017 112 Investigative Ophthalmology & Visual Science, January 1997, Vol. 38, No. 1 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933195/ on 05/05/2017 Pax-6 in Ocular Surface Epithelia 113 FIGURE l. Pax-6 expression in day 3 and day 5 chick embryos, detected by in situ hybridization and immunohistochemistry. (A,B) Heads of day 3 (HH stage 18) embryos were processed for wholemount in situ hybridization, followed by embedding in glycolmethacrylate and preparation of 10-^trn sections (A). Alternately, 14-/zm frozen sections were prepared, and immunocytochemistry with Pax-6 antibody followed (B). (top) Dorsal edge of optic cup is up. (left) Ocular surface to the. (C,D) Transverse frozen sections of a day 5 embryo were processed for Pax-6 mRNA by in situ hybridization (C) or immunohistochemistry (D). Orientation to that in A and B. (arrows) Surface ectoderm near edge of the future cornea! ectoderm. Scale bar = 64 /j,m (A,B); 128 ^m (C,D). of the developing eyelid (arrow). It appeared that all cells on the conjunctival side of the boundary expressed Pax-6, whereas none in the outer ectoderm of the eyelid were found to express the gene. Pax6 expression was observed to extend throughout the developing corneal ectoderm and conjunctiva. A puzzling observation in the day 5 chick embryo was that in situ hybridization failed to detect Pax-6 mRNA in the corneal and conjunctival epithelia (Fig. 1C, arrow), although signal was detected readily in the lens epithelium and retina of the same specimen. In contrast, the antibody detected high levels of immunoreacu've protein in the nuclei of these cells. Simi- larly, the nuclei of day 15 chick corneal epithelium showed strong signal with the antibody, but the cells gave no signal by in situ hybridization using RNA probes (not shown). This was surprising because generally we have found in situ hybridization to be a highly reliable technique. At first, we considered the possibility that the antibody detected another nuclear protein. If, on the other hand, the antibody detected genuine Pax-6 protein, we would have to conclude that the in situ hybridization method could not detect Pax-6 mRNA effectively in the day 5 cornea, even though there was no problem detecting mRNA in the lens and retina of the same specimen. Con 2. Pax-6 expression in day 6 chick embryos, detected by immunohistochemistry. Transverse vibratome sections (100 fjM) were prepared from anterior segments of chick embryo eyes and stained with Pax-6 antibody. (A) Low-magnification view of vertical section of (left to right); conjunctiva (con-arrow), underlying scleral mesenchyme, retinal pigment epithelium and retina. Dorsal is toward top. Arrow indicates edge of upper eyelid. At the lower edge, the retina and RPE are detached locally. Bright-field optics. (B) Higher magnification view of eyelid and conjunctiva in same specimen. Arrows indicate boundary of Pax6-expressing cells. Nomarski optics. Scale bar =100 //m (A); 50 fim (B). FIGURE Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933195/ on 05/05/2017 114 Investigative Ophthalmology & Visual Science, January 1997, Vol. 38, No. 1 1 2 3 4 5 kDa - 200 -116 -97 -66 46 ^ 37 • -45 • • -31 - 14 - 6.5 B -200 FIGURE 3. Western blot showing Pax-6 immunoreactive proteins in the chick embryo. Tissues were solubilized in electrophoresis sample buffer, and equal amounts of protein were loaded in each lane. (A) 5.8 ^g per lane. (B) 15.72 (j,g per lane. The lanes represent: (1) E15 corneal epithelium; (2) E15 (embryonic day 15 chick) retina; (3) E15 wing ectoderm; (4) E4 limb bud. Filters were incubated with Pax-6 antibody and visualized by ECL (Methods), with a 1-minute exposure time. Biotinylated molecular weight markers were visualized simultaneously by ECL to estimate molecular weight. Western Immunoblot Analysis of Pax-6 Expression in the Chick Embryo To resolve these issues, we dissected corneal ectoderm from day 15 chick embryos and conducted Western immunoblot analysis to determine whether the immunoreactive protein had the same apparent molecular weight as retinal Pax-6 (Figs. 3A, 3B). We found that a major band was strongly expressed in equivalent amounts in cornea (lane 1) and retina (lane 2). This band was absent in skin (lane 3) and day 4 limb bud (lane 4), tissues that do not express significant amounts of Pax-6 mRNA.7 The presence of this band was highly specific for the ocular tissues because it was not observed in skin and limb after increasing the sensitivity of detection (Fig. 3B). Comparison with markers (lane 5) determined its apparent molecular weight to be 46 kDa. There was a minor band at 37 kDa that was found primarily in retina and at lower levels in cornea. The 46-kDa band closely matches that predicted for the full-length protein and that observed for Pax-6 of quail.36 Weaker bands, apparently unrelated to Pax-6, appeared at higher molecular weights in all the samples (small arrowheads). Analysis of Pax-6 Gene Expression by Reverse Transcription-Polyraerase Chain Reaction At this point, it was still possible that low levels of Pax-6 mRNA in the corneal epithelium might generate the Pax-6 protein observed by immunocytochemistry and on western blots. This could occur if Pax-6 mRNA were translated more efficiently in the corneal epithelium or if the protein was much more stable in these cells. To determine the relative levels of chicken Pax-6 mRNA in the corneal epithelium and retina, total RNA was extracted from thesetissues,reverse transcribed by priming with random oligonucleotides, and PCR amplified with primers specific for chicken Pax-6 (Fig. 4A). Three bands (a = -400 bp doublet, b = 227 bp, and c = 185 bp) were visible in the retina (Fig. 4A, lane 1) and the corneal epithelium (lane 2). All three bands were completely absent in skin (lane 3), limb (Fig. 4A, lane 4), and a control reaction lacking cDNA template (lane 5). The 400-bp doublet indicated by band a is smaller than the 1-kb amplimer expected from genomic DNA35 and may consist of minor splice variants of Pax-6. Band c corresponds to the predicted 185-bp size for the canonical splice form of chicken17 and quail35 Pax-6. Band b corresponds to the 227-bp amplimer fragment expected from a splice form containing the 42-bp 4a exon35 (5a, in the mouse and human Pax-6 nomenclature37) in which 14 additional amino acids are inserted into the paired domain of the protein. The canonical and 4a splice forms of these mRNAs encode proteins close enough in size that they run as a single band in the gel (shown in Fig. 3). We have isolated and sequenced a chicken cDNA clone corresponding to this 4a mRNA splice form, and it generates an amplimer of the predicted size (data not shown). When band intensities were compared, die 4a splice variant appeared to be the predominant one in retina, but it was less abundant in cornea. To quantitate the relative amounts of these mRNA species, the RT-PCR reactions were carried out with radioactive nucleotides for a limited number of cycles. Relative molar yields of the excised bands are shown in Table 1 (see Methods) and an autoradiogram in Figure 4B. Similar amounts of total Pax-6 mRNA appear to be present in retina and cornea, making it clear diat we do not have to postulate any differences in trans- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933195/ on 05/05/2017 Pax-6 in Ocular Surface Epithelia 1 2 3 115 4 5 i c • Pax-6 gene expression in chick, analyzed by quantitative reverse transcription-polymerase chain reaction. (A) Total RNA was isolated from different chick embryo tissues and reverse transcribed, and cDNAs were normalized to the levels of 18S-rRNA. This was followed by 25 cycles of polymerase chain reaction (PCR) amplification and electrophoresis (see Methods). Lane 1 = E15 {embryonic day 15 chick) retina; lane 2 = E15 corneal epithelium; lane 3 = E15 wing ectoderm; lane 4 = E4 limb bud; lane 5 = PCR without cDNA template. Markers (PBR 322-MspI digest): 622, 527, 404, 307, 242 + 238, 217, 201, 190, 180, 160, 147, 123, 110 base pairs. The Pax-6-specific amplimer bands are: (a) ~400bp doublet corresponding to minor splice variants; (b) 227 bp: Pax-6 4a splice form, (c) 185 bp: Pax-6 canonical splice form. (B) Normalized cDNAs were PCR amplified for 16 cycles with 32P-dCTP, followed by electrophoresis and autoradiography. The format is the same as in A. the mouse. The mouse has a smaller eye that is sectioned easily in its entirety because it lacks the tough scleral ossicles found in the late embryonic and adult chick28 The eye of a 30-day mouse was removed with eyelids attached to the globe and was sectioned in the transverse plane. Pax-6 protein was detected in the nuclei of cells throughout the epithelial layer of the ocular surface, including the cornea, limbus, and entire conjunctiva. This domain extends into the entire palpebral epithelium of the eyelids, as shown in the section diagram (Fig. 5 I). A closer view of various regions (Fig. 5 II), showed that the nuclei of apparently all cells of the corneal epithelium contained Pax6 protein (Fig. 5 IIA). There did not appear to be any change in the intensity of signal or the frequency of expressing cells in the transition from cornea to the limbus and conjunctiva (Fig. 5 IIB). Close to the outer margin of the palpebral conjunctiva, the intensity of Pax-6 signal was seen to decrease, ending at a boundary defined by the hairs of the eyelid. The same abrupt boundary was observed for upper and lower eyelids (not shown). Among other tissues showing Pax-6 positive nuclei are the iris and ciliary body (Fig. 5 IIB), in which apparently all cells express the protein. Pax-6 antigen was expressed strongly in the anterior epithelium and bow region of the lens, as well as in the ganglion and amacrine layers of the retina (Figs. 5 I, 5 IIB). FIGURE 4. lational efficiency or protein stability to explain the high level of Pax-6 protein in the cornea. Both major splice forms contain the same carboxy terminus and, therefore, should be recognized by the antibody. Although overall levels are similar, the corneal epithelium and retina of the day 15 chick embryo have different ratios of the canonical and 5a splice forms. Pax-6 in the Adult Mouse Eye and Its Adnexal Tissues To determine the expression pattern of Pax-6 in postembryonic stages, it was more convenient to examine Pax-6 in the Anterior Segment of the Monkey Pax-6 expression was examined in sections of the anterior segment of an adult cynomolgus monkey. In monkey (Fig. 5 IID), the corneal epithelium was stained in a pattern similar to mouse, with all other regions of the cornea, including the stroma, completely negative for Pax-6. The signal was weaker than in the mouse, possibly because of differences in age, species differences, or tissue preparation. With the Pax-6 anti- i. Relative Abundance of Pax-6 mRNA Species in the 15-Day Chick Embryo* TABLE Amplimer (size) Retina (yield) Cornea (yield) 404 bp 227 bp (4a Pax-6) 185 bp (canonical Pax-6) Total Pax-6 mRNA 0.024 ± 0.004 0.796 ± 0.106 0.180 ± 4.0 1.00 0.026 ± 1.2 0.026 ± 4.8 1.092 ± 15.6 1.14 * Values represent the mean of triplicates ± standard error (SE). Pax-6 cDNA was amplified and radiolabeled with S2P dCTP in a 16-cyde PCR, followed by electrophoresis (see Fig. 4B), excision, and scintillation counting. Control samples without cDNA provided background counts, which were subtracted From all samples. Each cDNA sample was normalized to 18S rRNA, and the relative molar yield of each amplimer band was obtained. Relative molar yields for each amplimer band are finally normalized to the total Pax-6 mRNA found in retina, Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933195/ on 05/05/2017 116 Investigative Ophthalmology & Visual Science, January 1997, Vol. 38, No. 1 -4 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933195/ on 05/05/2017 Pax-6 in Ocular Surface Epithelia 117 FIGURE 5. Localization of Pax-6 protein in ocular tissues of mouse and monkey by immunohistochemistry. Vertical cryosections (14 /xm) were prepared from whole eyes and eyelids of a 30-day-old mouse and visualized with Pax-6 antibody and Vector VIP immunoperoxidase histochemistry. Panel I is a diagram summarizing Pax-6 distribution in the whole eye, and panel II shows higher magnification micrographs of boxed regions in the diagram. Dorsal is toward the top, and the exterior of the eye is toward the left. Micrographs show (A) palpebral conjunctiva and edge of eyelid, (B) fornix, and (C) cornea of mouse. (D) Vertical 10-fjim cryosection of adult cynomolgous monkey cornea, stained for Pax-6 as before, con = conjunctiva; ce = corneal epithelium; cb = ciliary body; h = hair; s = skin. Scale bar = 263 /urn (panel 1); 66 /zm (panel 2). body, signal was localized clearly in the epithelial cell nuclei. In sections incubated with rabbit preimmune serum to control for immunoglobin binding to the cornea, background signal was distributed throughout the specimen, in the cytoplasm, and in the nucleus (not shown). DISCUSSION Pax-6 is known to play an essential role in the establishment and growth of the vertebrate lens.10'12'15 We have found that the gene is expressed prominently in the early cellular precursors of the cornea and conjunctiva,8'1617 which are located in the periphery of the lens placode.2 The fact that human aniridia often is accompanied by adult corneal abnormalities13 has raised the question of whether human PAX6 has a functional role later in life and continues to be expressed at significant levels in the ocular surface. Mutations in Pax-6 have been found to be associated with other disorders not normally classified as aniridia but that share some features of the disease. Among these is autosomal dominant keratitis,38 in which corneal pannus formation is observed but without iris hypoplasia. Also associated with mutations in PAX6 is Peter's anomaly,39 a malformation of the cornea and anterior segment thought to result from a defect in the separation of the lens vesicle from the corneal epithelium.2 Abnormalities of the cornea and the ocular surface are a common feature of these "non-aniridia" PAX6 disorders, again suggesting the importance of PAX6 in the proper formation and function of these structures. The recent availability of antiserum specific for Pax-6 protein has allowed us to examine the expression of this gene in the late embryonic and mature ocular surface. This had not been feasible in the chick because in situ hybridization did not detect Pax-6 mRNA in the differentiated corneal ectoderm, although the technique readily detects mRNA in lens and retina. The reasons for this are unknown; in situ hybridization studies of the cornea, therefore, should be treated with caution. Our results with an antibody directed to the carboxy terminus of Pax-6 indicate that genuine Pax-6 protein is produced by the corneal epithelium at levels comparable to those in the retina. Further, RT-PCR with Pax-6-specific primers suggests that the levels of total Pax-6 mRNA in the corneal epithelium are similar to those in the retina. As far as we can determine, the corneal stroma and endothelium do not express Pax-6. The failure of in situ hybridization probes to detect Pax-6 mRNA in the day 5 chick embryo corneal epithelium is apparently a more general phenomenon because beta-actin mRNA is not detected efficiently in this tissue (data not shown). The cornea expresses both major splice forms normally found in the retina, the canonical Pax-68'9 and the 4a splice form35 (5a, mouse-human nomenclature37). The 4a form introduces an additional exon that inserts 14 conserved amino acids into the paired domain.35 Day 15 corneal ectoderm primarily produces the canonical form and much less of the 4a form, which is the predominant form in the day 15 retina. Earlier, when the retina undergoes rapid growth on embryonic days 4 and 5, the retinal assortment of these two Pax-6 splice forms is roughly 80% canonical and 20% 4a form, much closer to the distribution found in cornea (data not shown). The ratios of splice forms can vary with developmental stage and are not specific to tissue type. The paired domain of the 4a counterpart (5a) of human Pax-6 protein has a different DNA-binding specificity than the canonical form,37 suggesting that it may act on a different set of target genes. The relative expression levels of these two Pax-6 proteins may be of functional significance. Previous studies17 have shown that Pax-6 expression in the head ectoderm is distributed broadly in the neural-fold and neural-plate embryos. At later stages, Pax-6 expression becomes confined to two comet-shaped patches next to the optic vesicles (day 1.5 to 2 embryo). At this stage, the boundaries of these patches of Pax-6-positive cells are not sharply defined and show extensive intermixing of expressing and nonexpressing cells. In the day 4 and 5 chick embryos, the center of this patch of tissue has already invaginated to form the lens. The peripheral Pax-6-expressing cells remain on the surface and, at their edges, still are intermixed extensively with nonexpressing Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933195/ on 05/05/2017 118 Investigative Ophthalmology & Visual Science, January 1997, Vol. 38, No. 1 cells. By day 6, however, when the eyelids begin to appear, the boundary between Pax-6 expressing and nonexpressing cells becomes precisely defined at the edge of the eyelid. This progressive narrowing and sharpening of the Pax-6 domains, which happens as morphologic features appear, follows a general theme observed for other homeobox genes involved in regulating the development of pattern.3'6'31 It should be noted that Pax-6-expressing cells are located on the interior surface of the eyelid but are not found in the mesoderm or skin, suggesting that the formation of these structures might not require function of the gene. This is consistent with the presence of external eyelid features on anophthalmic mouse11 and human14 newborns that completely lack a functional PAX6 gene. In addition to its presence during development, Pax-6 protein continues to be expressed in cells of the adult ocular surface. In the corneal epithelium, a highly specialized tissue designed to maintain a smooth and transparent surface for the eye, Pax-6 might be involved in regulating the expression of genes that determine its cellular structure and composition. Because there is evidence that Pax-6 direcdy regulates the expression of crystallin genes in the lens22"24 and it is known that the corneal epithelium contains crystallin-like proteins,40 Pax-6 might regulate the expression of such crystallin-like proteins in the adult cornea. We were surprised, however, to find that Pax-6 expression is not limited to the cornea but that the entire conjunctival ectoderm expresses the gene, all the way to the edge of the eyelids. This suggests that Pax-6 may be required for maintaining adult function of the conjunctiva, possibly the secretory functions in this mucosal ectoderm. It is interesting that in the embryonic and adult olfactory epithelium, the mucosal supporting cells express Pax-6. They are the only other ectodermally derived cells in the body to express and require Pax-6 for their development.812'16 In the three species we have examined, Pax-6 is expressed in the corneal epithelium but not in the stroma, an observation that reflects the fundamentally different origins of these tissues. Conjunctiva and corneal epithelium are derived from the surface ectoderm of the head, whereas stroma and endothelium arise from neural crest cells.2'41 Pax-6 expression has not been observed in neural crest or mesodermal cells of the mouse or chick embryo and appears restricted to specific regions of neural, ectodermal, and endodermal tissue.8'17 Although detailed features of the adult iris are not readily visible in Figure 5 because of heavy pigmentation, the pattern of Pax-6 expression appears to follow the same general rule in this structure. The vertebrate iris has a substantial contribution from neural crest, which generates the stromal cells. In the newly hatched chick, these stromal cells do not express Pax-6 protein, whereas the ectodermally derived posterior iris epithelium clearly expresses the gene. Islands of cells in the stroma that appear to correspond to the iris sphincter and dilator muscles express Pax-6. If confirmed, this observation again would be consistent with the atypical origin of these muscles, which derive from the neural ectoderm of the optic vesicle.41 Evidence of a close functional relationship between corneal and conjunctival cells is found in the remarkable interaction of these two tissues during the healing of extensive corneal wounds.42"45 Normally, cells lost from the corneal surface are replaced by proliferating cells that originate in a slowly cycling stem cell population located in the limbus at the corneal margin.46 When limbal stem cells are destroyed, the corneal epithelium is replaced by an influx of cells migrating from the conjunctiva.45'47"49 To a surprising degree, these migrating conjunctival cells can take on the role of corneal epithelium, and it has been suggested that they have the capability to transdifferentiate fully into this tissue.43'30 However, the persistence of goblet secretory cells and other features indicate that this transdifferentiation is incomplete,45'31 although the conjunctival cells can acquire the essentials of corneal structure and function. The expression of Pax-6 in both these tissues may reflect their close similarity as ocular structures and possibly serve to maintain the expression of genes that define their cellular identity. A final issue concerns ocular abnormalities observed in patients with aniridia. In cases of aniridia associated with heterozygous mutations in Pax-6, it is thought that the disorder is caused by a decrease in the functional gene dosage from two copies to one. 91014 In early adulthood, an opaque and vascularized region of the cornea known as a pannus often develops in these individuals, and there have been few clues concerning how it arises.13 Recently, the ocular surfaces of patients with aniridia were visualized by impression cytology, and considerable numbers of conjunctival goblet cells were found embedded in the corneal epithelium.52 The origin of these out-of-place cells is thought to be an influx of partially transdifferentiated conjunctival cells populating the surface of the cornea. In addition, the limbal structures that contain the corneal stem cells, known as the Palisades of Vogt,33 were absent or greatly reduced, suggesting that corneal stem cell deficiency might be the cause of conjunctival invasion of the cornea, very much along the paradigm described earlier for wound healing. We have shown that mouse Pax-6 protein is expressed at high levels in adult limbal cells, indicating that it probably has the opportunity of time and place to be involved direcdy in the corneal phenotype of patients with aniridia. Together, these observations raise the possibility that high-level expression of Pax-6 is necessary for the maintenance or proliferation of corneal Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933195/ on 05/05/2017 Pax-6 in Ocular Surface Epithelia stem cells. This notion is supported by the observation that when mouse Pax-6 protein is expressed from a powerful promoter in NIH 3T3 fibroblasts, it can induce proliferation strikingly in the manner of an oncogene. 54 The conjunctiva might be dependent on Pax6 for the maintenance of its stem cells, but it appears that this function is less sensitive to a decrease in Pax6 gene dosage. It is possible, however, that careful examination of individuals with aniridia will reveal abnormalities in conjunctival structure or function. 119 12. 13. 14. Key Words conjunctiva, cornea, homeobox, Pax-6, vertebrate 15. Acknowledgments The authors thank Drs. Janine Davis and Randall Reed of the Howard Hughes Medical Institute for the gift of Pax-6 antiserum, Wing Fun Lau, and Dr. Ruben Adler for the mice, and Dr. Harry Quigley for the monkey anterior segments. They also thank Dr. Morton Goldberg for valuable discussions and Dr. Neil Delia for insightful comments on the manuscript. References 16. 17. 18. 1. 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