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A.J. Quantock, E.C. Gealy, B.C. Kerr, A.J. Hayes, C.E. Hughes, R.D. Young, B. Caterson, J.R. Ralphs; Differential Expression of Keratocan in the Developing Chick Cornea With the Onset of Transparency . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3005.
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Keratocan is a small leucine–rich proteoglycan that is substituted with keratan sulphate glycosaminogycan chains. It is expressed in the cornea, and is believed to play a key role in corneal transparency. The aim of this study is to determine the onset and localisation of keratocan expression in the developing chick cornea by using a monoclonal antibody (KER–1) which recognises a protein epitope on the keratocan core protein.
Chick corneas (E10–E18) were mounted in OCT, frozen with dry ice and cryosectioned at 10µm. Cryosections were treated with 0.4U/ml keratanase, 0.004U/ml keratanase II, 0.4U/ml Chondroitinase ABC and 0.002U/ml endo–beta galactosidase for 1 hour at 37°C and labelled by indirect immunofluorescence with monoclonal antibody KER–1 followed by Alexa Fluor 488 conjugated goat anti–mouse IgG secondary antibody. Sections were mounted in Vectashield containing DAPI and analysed using an Olympus BX61 microscope and F–View digital camera or Leica SP2 AOBS confocal microscope.
Keratocan was detected at all stages of chick corneal development investigated (E10–E18). However, distinct differences in localisation and organisation were observed between early (E10, E12, E13) and later (E14, E15, E18) stages of development where the onset of transparency occurs. At early stages, keratocan appeared both cell associated and in the extracellular matrix of the stroma and was particularly concentrated in the anterior region. In contrast, by E14 keratocan was organised extracellularly in long fibrillar arrays, presumably associated along the collagen fibrils within the stromal matrix. This fibrillar label was concentrated in the superficial stroma, and persisted through E15 and E18. Bowman’s layer was unlabelled at all stages. Keratocan was also present at cell–cell interfaces in the corneal epithelium at later stages of development.
Keratocan expression at the protein level is concentrated in the anterior stroma of the developing chick cornea. In this tissue region a major structural reorganisation of keratocan occurs between E13 and E14. Keratocan is associated with control of fibril diameter and spacing in corneal stroma, factors crucial to transparency. The timing of the structural reorganisation of keratocan anteriorly is consistent with compaction and onset of transparency in the anterior chick cornea, and emphasises the potential importance of keratocan expression in corneal development.
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