While tissue form and function is highly dependent upon tissue-specific collagen composition and organization, little is known of the mechanisms controlling the bundling of collagen fibrils into fibers and larger structural designs that lead to the formation of bones, tendons and other tissues. Using the cornea as a model system, our previous 3 dimensional mapping of collagen fiber organization has demonstrated that macrostructural organization of collagen fibers involving interweaving, branching and anastomosing plays a critical role in controlling mechanical stiffness, corneal shape and refractive power. In this work, the cellular and mechanical mechanisms regulating critical events in the assembly of collagen macrostructure are analysed in the developing chicken cornea. We elucidated the temporal events leading to adult corneal structure and determined the effects of intraocular pressure (IOP) on the organization of the collagen macrostructure. Our findings indicate that the complex adult collagen organization begins to appear on embryonic day 10 (E10) after deposition of the primary stroma and full invasion of keratocytes. Importantly, organizational changes in keratocytes appearing at E9 preceded and predicted later changes in collagen organization. Corneal collagen organization remained unaffected when the development of IOP was blocked at E4. These findings support a primary role for keratocytes in controlling stromal organization, mechanical stiffness and corneal shape that are not regulated by the IOP. Our findings also suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function.

Statement of significance

This work by using an ex ovo model system, begins to investigate the potential mechanisms controlling collagen fibril macrostructure. In particular, this work highlights a convergent role for the corneal keratocytes in organizing the complex collagen macrostructure, necessary to support high visual acuity. Our data supports that the intraocular pressure does not influence collagen fibril macrostructure and suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function. Clearly understanding the cellular and molecular mechanisms that underlie collagen fibril macrostructure will be highly beneficial for future tissue engineering and regenerative medicine applications.

In this review, we discuss current methods for studying ocular extracellular matrix (ECM) assembly from the 'nano' to the 'macro' levels of hierarchical organization. Since collagen is the major structural protein in the eye, providing mechanical strength and controlling ocular shape, the methods presented focus on understanding the molecular assembly of collagen at the nanometre level using X-ray scattering through to the millimetre to centimetre level using non-linear optical (NLO) imaging of second harmonic generated (SHG) signals. Three-dimensional analysis of ECM structure is also discussed, including electron tomography, serial block face scanning electron microscopy (SBF-SEM) and digital image reconstruction. Techniques to detect non-collagenous structural components of the ECM are also presented, and these include immunoelectron microscopy and staining with cationic dyes. Together, these various approaches are providing new insights into the structural blueprint of the ocular ECM, and in particular that of the cornea, which impacts upon our current understanding of the control of corneal shape, pathogenic mechanisms underlying ectatic disorders of the cornea and the potential for corneal tissue engineering.

The meibomian and sebaceous glands secrete lipids to prevent desiccation of the ocular surface and skin, respectively. Precisely how these holocrine tissues regenerate is not well understood. To address this, we characterized keratin 5(+) (K5) label-retaining cells (LRCs) and the lineage tracing of keratin 14 (K14) progenitors in mouse meibomian glands. Using the tet-off H2B-GFP/K5tTA mouse, H2B-GFP fluorescence dilutes 2-fold with every division in K5(+) cell nuclei after doxycycline administration. In 3D reconstructions generated over a >28-day doxycycline chase, we observed LRCs at the acinus entrance where K6(+) ductal epithelium terminates. For lineage tracing, K14CreER(T2)-Confetti mice were injected intraperitoneally with tamoxifen and euthanized at 23 and 59 weeks later. Meibomian gland acini in these mice were either monochromatic or dual-colored, whereas the duct exhibited multiple colors. In conclusion, LRCs are likely to direct meibomian gland turnover and may exist as two distinct unipotent progenitors that renew ductal and acinar tissue separately.