Abstract
Purpose: :
Sulfation of collagen fibril–associated glycosaminoglycans (GAGs) substituted on keratan sulfate (KS) proteoglycans (PGs) is thought to be essential for transparency of the corneal stroma. We used sulfation motif–specific antibodies to identify the sequence of appearance of sulfated KS GAG at light and electron microscopic levels during development of the chick cornea.
Methods: :
Corneas were excised from chick embryos every 2 days from day 8 through day 18 of incubation and either frozen unfixed in OCT for immunofluorescence microscopy, or mildly fixed for 1h in 4% paraformaldehyde or 2% paraformaldehyde/0.05% glutaraldehyde, then embedded in Lowicryl K4M resin at –25°C for immunoelectron microscopy. Sections, without or with predigestion with chondroitinase ABC, were labelled with monoclonal antibodies 5D4 or 1B4 which react with high and lower sulfated epitopes, respectively, on KS GAGs. Antibody positive sites were detected by immunofluorescence microscopy with secondary antibodies conjugated to Alexafluor 488, or 10nm gold for electron microscopy.
Results: :
Sulfated KS GAG epitopes were first identified by 5D4 and 1B4 in embryo corneas at 10 days of incubation, appearing closely associated with collagen fibrils in the extracellular matrix and at keratocyte cell surfaces. 5D4 KS labelling appeared concomitantly with bundles of regularly–arranged collagen fibrils, the rudiments of presumptive lamellae. Labelling was more concentrated in the anterior stroma at day 12 through 14 of incubation, but subsequently became more widespread and was uniformly distributed throughout the full stromal thickness by day 18. Localisation of the 1B4 KS epitope accompanied that for 5D4, but at less intense levels.
Conclusions: :
High and lower sulfated KS GAG epitopes were detected using anti–KS sulfation motif–specific antibodies as early as day 10 of incubation in embryonic chick corneas. This coincides spatially and temporally with the appearance of regularly arranged collagen fibril bundles in the early stages of the orthogonal lamellar organization of the corneal stroma.
Keywords: cornea: stroma and keratocytes • proteoglycans/glycosaminoglycans • immunohistochemistry