Abstract
Purpose::
In chick corneal development, keratocytes invade the primary stroma and lay down the highly ordered collagenous secondary stroma. The aim of the present study is to investigate the role of the cytoskeleton in oriented collagen secretion and to observe the fates of the C-and N termini of type I procollagen as matrix is deposited.
Methods::
Chick embryo corneas (E8-18) were frozen in OCT compound on dry ice and cryosectioned. Sections were labelled for actin using phalloidin-AlexaFluor 488 and for type I procollagen using monoclonal antibodies M38 and SP1D8 (which recognise the carboxyterminal and aminoterminal propeptides of type I collagen respectively) and Alexa Fluor 594 goat anti-mouse IgG. Specimens were mounted in Vectashield/DAPI and examined with Olympus BX61 and Leica SP2 AOBS microscopes.
Results::
Prominent arrays of actin fibres were present in keratocytes throughout the developmental period. In approriate section planes they could be seen to form orthogonal arrays matching the matrix orientation of the tissue - cells were oriented with the collagenous lamellae. Antibody M38 labelled intracellularly, showing small bright foci within cells, colocalising in rows along the actin fibres. At early stages SP1D8 gave a similar distribution, however from 10 days onwards this label was clearly associated with the extracellular matrix as well as being intracellular along the actin fibres. Comparison between SP1D8 label and DIC images which showed collagen fibre bundles revealed an exact coincidence between bundles and SP1D8 label. In addition, Bowmans layer was prominently labelled with this antibody from early in development, whereas M38 label was generally at a low level, with a prominent, transient burst of particulate label there at day 14.
Conclusions::
These results reinforce our previous studies suggesting that the actin cytoskeleton has an important role in cell orientation and collagen trafficking during the deposition of the highly organised chick secondary stroma. They also show different processing of the C and N termini of type I procollagen during the secretory process. Both can be detected intracellularly, but on secretion label for the C terminus disappears, suggesting its rapid degradation, whereas label for the N terminus is retained in the stroma, suggesting that it is inorporated in the extracellular matrix. It also appears to become a prominent component of Bowman’s layer.
Keywords: cornea: stroma and keratocytes • cytoskeleton • extracellular matrix