Collagen Secretion Is Associated With the Actin Cytoskeleton in the Developing Chick Cornea

Abstract

Purpose: : Mechanisms of early matrix orientation in the developing corneal stroma are unclear. In a structurally similar tissue, the annulus fibrosus of the intervertebral disc, lamellar deposition is preceded by the formation of sheets of parallel fibroblasts, oriented by cell–cell interactions and prominent actin stress fibres. The aim of this study is to investigate the role of actin fibres in controlling matrix deposition during corneal development and their association with collagen secretory pathways.

Methods: : Chick embryo corneas (E8–E18) were either frozen unfixed in OCT and cryosectioned or treated with trypsin–EDTA and 0.15% collagenase to isolate stromal keratocytes for monolayer culture. Sections or cultures were labelled for actin using phalloidin–FITC and/or for type I procollagen using monoclonal antibody M–38 (which recognises the carboxyterminal propeptide of type I collagen) and Alexa Fluor 488 or 594 goat anti–mouse IgG secondary antibodies. Specimens were mounted in Vectashield/DAPI and examined with an Olympus BX61 microscope and F–View digital camera or Leica SP2 AOBS confocal microscope.

Results: : Actin fibres were detected running along the long axis of stromal keratocytes at developmental stages E8–E18. Furthermore, an orthogonal arrangement of actin fibres suggested that cells in different layers of the stroma were orientated at different angles. Type I procollagen was detected as bright intracellular speckles, with speckles often observed aligned in rows within the same cell. Dual labelling demonstrated a strong co–localisation of procollagen with actin stress fibres, confirmed by both conventional fluorescence microscopy and confocal microscopy. Prominent actin fibres and intracellular type I procollagen were detected in cultured stromal keratocytes, with fibres within the same cell being aligned in a parallel fashion; however, these did not co–localise with the procollagen.

Conclusions: : Actin fibres are present during development of the chick corneal stroma and their orthogonal arrangement suggests a potential role in cell and matrix organisation in the stroma. Furthermore, co–localisation of intracellular type I procollagen with the actin cytoskeleton suggests that actin fibres may be involved in collagen trafficking and secretion. In vitro, experiments indicate that cells in monolayer do not secrete collagen in the same way as cells in vivo.

Keywords: cornea: stroma and keratocytes • cytoskeleton • immunohistochemistry