ARTICLE |
Correspondence to: Yoshihiro Akimoto, Dept. of Anatomy, Kyorin U. School of Medicine, Mitaka, Tokyo 181-8611 Japan. E-mail: yakimoto@kyorin-u.ac.jp
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Summary |
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The expression and distribution of the long form of Type XII collagen were investigated histochemically during chicken corneal development using a monoclonal antibody (P3D11) raised against the N-terminal domain of chicken Type XII collagen. Specificity of the antibody was confirmed by immunoprecipitation before and after bacterial collagenase digestion. Immunofluorescent microscopic studies showed that during chicken cornea formation, the long form of Type XII collagen is initially detected on Day 3 embryo (stage 19) in the sub-epithelial matrix of the corneal periphery and in the matrix around the optic cup. On Day 5 embryo (stage 27) the long form was expressed in the primary stroma. Thereafter, as the secondary stroma was formed, the long form localized in the sub-epithelial and sub-endothelial matrices and in the anterior region of the limbus (corneoscleral junction) before the formation of Descemet's and Bowman's membranes. After hatching, the immunoreactivity decreased predominantly in the sub-epithelial and sub-endothelial matrices but remained at the anterior region of the limbus. Immunoelectron microscopic examination demonstrated that the long form localizes in the Descemet's and Bowman's membranes and along the collagen fibrils in the stroma with a periodic repeat. Based on the distribution of the long form of Type XII collagen in the sub-epithelial and sub-endothelial matrices and limbus, it was suggested that the long form of Type XII collagen is involved in formation of the Descemet's and Bowman's membranes and in stabilization of the limbus. (J Histochem Cytochem 50:851862, 2002)
Key Words: type XII collagen, corneal development, extracellular matrix, Descemet's membrane, Bowman's membrane, limbus
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Introduction |
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TYPE XII collagen belongs to a group of fibril-associated collagens with interrupted triple helices (FACITs), together with Types IX and XIV collagen molecules (
The cornea consists of five layers: corneal epithelium, Bowman's membrane, corneal stroma, Descemet's membrane, and corneal endothelium. The corneal epithelium consists of non-keratinized stratified squamous cells and overlies Bowman's membrane. The Bowman's membrane consists of homogenous lamina and is composed of randomly oriented collagen fibrils (1820 nm in diameter). The corneal stroma consists of parallel bundles of collagen fibrils (2325 nm in diameter). Descemet's membrane is a basement membrane underlying the corneal endothelium. The corneal endothelium is a single layer of flattened cells that face towards the anterior chamber in the eyeball. In adult human cornea, the long form is present in the corneal stroma and Bowman's membrane but is absent in the limbus (
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Materials and Methods |
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Materials
Tissue culture medium, fetal calf serum, chicken serum, and other tissue culture supplies were obtained from Gibco BRL (Grand Island, NY). Highly purified collagenase from Clostridium histolyticum was obtained from Worthington Biochemicals (Lakewood, NJ). Protein ASepharose 4FF, [35S]-methionine (1000 Ci/mM), and Ni-chelate affinity absorbent (Ni-NTA-agarose) were purchased from Amersham Life Science (Arlington Heights, IL) and Qiagen (Hilden, Germany), respectively.
Tissues
All experimental procedures using laboratory animals were approved by the Animal Care and Use Committee of Kyorin University School of Medicine. Fertilized eggs of White Leghorn chickens were incubated at 38C until desired stages. The embryonic stages were classified according to the nomenclature of
Expression of Recombinant Chicken Type XII Collagen
The cDNA encoding the P3 fragment of the N-terminal region in chicken Type XII collagen (
Metabolic Labeling of Fibroblasts with [35S]-Methionine
Chicken embryonic fibroblasts (CEFs) prepared by the method described previously (
Generation of Monoclonal Antibody
Approximately 200 mg of recombinant chicken Type XII collagen dissolved in PBS was mixed with complete Freund's adjuvant and injected IP into BALB/c mice. Two booster injections with the same amount of the antigen were given at 2-week intervals. On the third day after the final booster injection, the spleens were excised and isolated splenocytes were fused with SP2/0 myeloma cells using 50% polyethylene glycol 1500 (BoehringerManheim; Manheim, Germany) according to the manufacturer's instructions. Hybridomas growing in HAT selection medium were initially screened by immunohistochemical study using chicken tendon tissue sections and then by immunoprecipitation using the [35S]-Met-labeled proteins. The monoclonal antibody obtained in the present study was designated as P3D11.
Immunoprecipitation of Type XII Collagen
CEFs metabolically labeled with [35S]-Met were washed four times with serum-free DMEM and lysed with 1 ml of PBS containing 2% Triton X-100, 1 mM EDTA, and 3 mM phenylmethylsulfonylfluoride (PMSF) (lysis buffer) on ice for 10 min. The following procedures were performed at 4C unless otherwise specified. The cell lysates were centrifuged at 10,000 x g for 30 min. The resultant supernatant was incubated with the antibody (2 mg/ml) for 15 min, and the immune complexes were mixed with protein ASepharose beads (20 µl gel for 1 ml of cell lysates). The immunocomplexes bound to the beads were washed several times with the lysis buffer and with Dulbecco's PBS, and then eluted from the beads with 100 mM acetic acid or by boiling in 1% SDS. In some experiments the immunoprecipitated Type XII collagen was digested with bacterial collagenase (0.1 U/ml in DPBS) at 37C for 60 min. The reaction was terminated by adding SDS-PAGE sample buffer containing 1 mM dithiothreitol (DTT), boiling for 90 sec, and then subjection to SDS-PAGE.
Immunofluorescent Light Microscopic Observation
Whole chicken embryos or eyeballs were embedded in OCT compound (Miles; Elkhart, IL) and frozen with liquid nitrogen. Sections (10 µm thick) were made with a cryostat (Sakura Seiki; Tokyo, Japan), placed on egg albumin-coated slides, and fixed with 2% formaldehydePBS at 4C for 30 min. After washing with PBS, nonspecific binding sites were blocked by incubation with 5% donkey serum for 30 min. Sections were then incubated with P3D11 antibody or with non-immunized mouse IgG for 1 hr at room temperature, washed with PBS, and subsequently incubated for 1 hr with Cy3-conjugated donkey anti-mouse IgG antibody (Jackson Immunoresearch; West Grove, PA). Some sections were further incubated with fluorescein-labeled phalloidin (Molecular Probe; Eugene, OR) to stain F-actin. Fluoresceinphalloidin strongly stained the ciliary muscle and also delineated the cell border, which aided identification of immunopositive sites. Nuclei were stained with 4,6-diamidine-2-phenylindole hydrochloride (DAPI) (BoehringerMannheim). After washing with PBS, the specimens were mounted in 90% glycerol0.1 M Tris-HCl buffer (pH 8.5) containing 0.5 mM p-phenylene diamine, which prevents fading of fluorescence during microscopic examination, and observed under an epifluorescent microscope (Nikon; Tokyo, Japan) equipped with chilled color CCD camera Type C5985 (Hamamatsu Photonics; Hamamatsu, Japan).
Immunoelectron Microscopic Study
The HRP labeling study was carried out as described previously (
For the colloidal gold labeling study, the HRP-conjugated secondary antibody was replaced by nanogold (Nanoprobes; Stony Brook, NY). The specimens were washed with PBS, then fixed in 2% glutaraldehyde. After rinsing with deionized water, silver enhancement of nanogold was carried out with HQ silver (Nanoprobes) according to the manufacturer's protocol. Development time was 3 min. The specimens were rinsed with deionized water, then fixed with 0.1% OsO4 for 30 min, dehydrated through a graded series of ethanol solutions, exposed to propylene oxide, and embedded in Epon 812. Ultrathin sections were stained with lead citrate and uranyl acetate and observed under a transmission electron microscope (JEM-1010). In some experiments, samples were fixed in 2.5% glutaraldehyde and processed for conventional EM observation as described previously (
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Results |
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Specificity of Monoclonal Antibody Against Chicken Tendon Type XII Collagen
Occurrence of the long and short forms in Type XII collagen was reported in several animal species (
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Immunohistochemical Observation of the Long Form of Type XII Collagen in Chicken Cornea by Light Microscopy
To elucidate when and where the long form of Type XII collagen is initially expressed and whether or not its expression changes in the cornea during chicken development, the localization of Type XII collagen in chicken cornea was examined immunohistochemically using the monoclonal antibody described above. The optic vesicles are formed on Day 1.5 of incubation (stage 10), and no immunoreactivity to Type XII collagen was detected in the optic vesicles and the lens placode (Fig 3A). After this stage the lens placode begins to invaginate, and on Day 3 (stage 19) the lens vesicle pinches off and the overlying ectoderm is induced to become corneal epithelium. At this stage the immunoreactivity was detected in the sub-epithelial matrix located peripherally in the cornea, and around the optic lip (Fig 3B3D). In particular, intense immunoreactivity was present around the outer surface of the optic cup at the proximal side (Fig 3D). By Day 4, the corneal epithelium secretes and forms the primary stroma, and the mesenchymal cells, which are destined to become the corneal endothelium, begin to invade the area. On Day 4 (stage 24), strong immunoreactivity was detected in the mesenchyme between the corneal epithelium and optic lip (Fig 3E and Fig 3F). Weak immunoreactivity was also present between the corneal epithelium and the lens (Fig 3E and Fig 3F). Endothelial cell migration is almost completed by Day 4.55 of incubation (stage 2526). On Day 5 of incubation (stage 27), the immunoreactivity was distributed throughout the primary stroma and in the mesenchyme (Fig 4A). After this stage, the immunoreactivity was localized in the sub-epithelial and sub-endothelial regions (Fig 4B4D). However, the immunoreactivity disappeared in the sub-epithelial region by Day 10, but not completely (Fig 4B). In contrast, after Day 10 of incubation, the immunoreactivity increased in the sub-epithelial region. On Day 14 of incubation the cornea begins to condense, becomes transparent, ceases its rapid growth, and becomes resistant to deformation (
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Immunohistochemical Observation of the Long Form of Type XII Collagen in Chicken Cornea by Electron Microscopy
To examine the fine localization of the long form of Type XII collagen in chicken corneal matrix, immunoelectron microscopic analysis was performed by an indirect method using the HRP-conjugated antibody. The HRP method exhibited similar distribution patterns of the long form of Type XII collagen to those revealed by immunofluorescence study as described above. The corneal epithelium laid down the primary corneal stroma on Day 3 and was located close to the optic cup (Fig 5A), and the basement membrane underlay the corneal epithelium and optic cup, in which collagen fibrils were sparsely located near the basement membrane in the stroma (Fig 5B). Immunoreactivity was detected in the basement membrane and along the collagen fibrils attached to the basement membrane, with a periodic repeat of 5560 nm in Day 3 chicken embryonic cornea (Fig 5C and Fig 5D). Descemet's membrane first appears subjacent to the corneal endothelium in Day 9 chick embryo, and Bowman's membrane appears subjacent to the corneal epithelium in Day 12 chick embryo (
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Discussion |
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Type XII collagen contains the C-terminal region in which the triple-helical domains (COL1 and COL2) are interrupted by non-triple-helical domains (NC1 and NC2) (
In earlier studies on Type XII collagen during chicken corneal formation (
Further studies on the localization of the short form are to be carried out by using the antibody specific to the form. Because the limbus is a transitional zone between the cornea and sclera and secretes aqueous humor, and because stem cells of the corneal epithelium are stored in the limbus, the long form of Type XII collagen expressed in the anterior region of the limbus could be involved in induction of the stem cells to differentiate and in adjustment of the tensile strength (
The long form of Type XII collagen also exists in the cornea of other animal species (
The present immunoelectron microscopic study revealed that the long form of Type XII collagen localizes in the basement membrane and along the collagen fibrils that are associated with basement membrane in chicken cornea. Interestingly, the long form of Type XII collagen was expressed periodically at 5560 nm intervals along the surface of bundled collagen fibrils. Similar results were also obtained in human and bovine tissues (
Because the N-terminal and C-terminal regions of Type XII collagen have been shown to interact with cells and extracellular matrix components, the generation of the splicing variants during the development and differentiation could contribute to dramatic changes in the macromolecular interactions and their arrangements in connective tissues, which is quite important for the tissue construction. Therefore, the temporal and spatial expression of the long form of Type XII collagen, as observed in the present study, imply its crucial roles in chicken eye formation by a yet unestablished mechanism.
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Acknowledgments |
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Supported by Grants-in-Aid for Scientific Research (10877004 to YA and 12680708 to KF) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
We wish to express our appreciation to Mr M. Fukuda, Ms S. Matsubara, Ms M. Kanai, and Ms T. Shibata in the Laboratory for Electron Microscopy and Department of Anatomy at Kyorin University School of Medicine for their technical assistance. We thank Dr A. Kudo for valuable discussions.
Received for publication August 26, 2001; accepted December 27, 2001.
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