Keratocyte‐mediated establishment of lamellar structure in corneal development

Abstract

Abstract Purpose Mechanisms by which presumptive keratocytes in the developing cornea engineer the 3‐dimensional lamellar matrix of the transparent stroma remain unknown. We used Volume Scanning Electron Microscopy to observe lamella formation in embryonic avian cornea in order to understand better corneal stromal biosynthesis, transparency and repair. Methods Corneas were obtained from chick embryos at E10 and E14, when a loose structure of collagen fibril bundles condenses into the lamellar organisation of the mature stroma. Aldehyde‐fixed corneas were contrast‐enhanced in osmium ferricyanide and tannic acid and embedded in Durcupan resin. Toluidine blue‐stained sections were used to locate suitable sites, subsequently examined in unstained ultrathin sections in a Jeol 1010 transmission electron microscope. The polished faces of the resin blocks were then imaged using a BSE detector in a Quanta 3D FEG FIB/SEM via an alternate slice‐and‐view protocol. 50nm slicing was performed with a focused gallium ion beam and image series were collected over 18h. Image stacks were processed using ImageJ software. Results Transmission and volume scanning electron microscopy reveal complex compartmentalisation of developing chick corneal stroma. Keratocyte filopodia extend many microns from cells into the extracellular space. These invest collagen fibrils, with varying orientations, and persist as the stroma condenses, appearing to orchestrate the spatial and directional organisation of the component lamellae. Conclusion Volume SEM reveals in 3D the complex cellular morphology of prospective keratocytes in developing chick cornea with extensive filopodia, which presumably channel and direct collagen fibril bundles into an emergent lamellar architecture.