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P.S. Howard, H. Maisenbacher, S. Decker, H. Yeh, P. Billings, J. Rosenbloom; Developmental Expression of Big-h3 in the Chick Cornea . Invest. Ophthalmol. Vis. Sci. 2003;44(13):865.
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Purpose: Big-h3 is an extracellular matrix protein found in many developing and mature tissues. Although the function of Big-h3 is unknown, mutations in Big-h3 have been shown to result in several forms of corneal dystrophies. Using the developing chick cornea as a model system, we sought to determine the expression of Big-h3 mRNA and protein throughout development. Methods: Developmentally staged chick eyes and/or corneas were microdissected and processed by whole mount in-situ hybridization (ISH) utilizing digoxigenin labeled chick sense and anti-sense Big-h3 RNA probes. For immunohistochemistry (IHC) and confocal microscopy, unfixed corneas (days 3-18) were cryosectioned (5-7 microns) and incubated with monospecific antibodies generated from sequence derived synthetic peptides to the amino or carboxy terminal regions of Big-h3. Results: Expression of Big-h3 mRNA in the developing cornea was detected at day 5 (stage 27) to day 6.5 (stage 30) in the region of invading peri-corneal mesenchymal cells, whereas no signal was detected at days 7, 9 and 17 in the cornea proper. In contrast, at day 3 (stage 20) localization of Big-h3 protein was confined to the corneal epithelium, and subsequently stained the endothelium and peri-corneal mesenchymal cells invading the primary stroma at day 5. Throughout all remaining embryonic stages, protein expression occurred within the cells of all corneal layers (epithelia, stromal keratocytes, endothelia). Staining for Big-h3 in the stromal matrix was minimal, indicating lack of secreted protein or masking of the epitopes. Treatment with dilute acetic acid to unmask epitopes resulted in exposure of very fine, filamentous fibers within the stromal matrix adjacent to collagen fibers. Conclusions: 1. Expression of Big-h3 mRNA occurs from embryonic days 5-6.5 (earliest days examined) in corneal endothelium and the peri-corneal mesenchymal cells invading the primary stroma. From days 8-18, Big-h3 mRNA is not expressed in corneal tissues as determined by ISH. 2. Big-h3 protein in corneal epithelium and endothelium is expressed early (days 3 and 5, respectively), and is maintained throughout development. 3. Big-h3 protein is associated with stromal keratocytes and fine filamentous matrix adjacent to collagen fibers in the stroma. These studies show that expression of Big-h3 begins early in ocular development and that the organization of the extracellular protein in the stromal matrix is complex.
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