A Comparative Study of Vertebrate Corneal Structure: The Evolution of a Refractive Lens

Vision is our primary means of sensing our environment, and in the vertebrate eye visual acuity is dependent, in part, on the quality of the eye's refractive components, namely the lens and the cornea. The cornea specifically fulfills dual roles, acting as both a protective transparent cover that encases the inner parts of the eye, and as a refractive lens that focuses light back to the neurosensory retina. In its refractive role, the cornea's shape directly and significantly influences visual acuity, as evidenced by the success of refractive surgical procedures that are based largely on mechanical reshaping the anterior corneal surface. Although corneal shape is thought to be determined by the structure and biomechanics, the mechanisms regulating shape remain largely unknown. Overall, the optical performance of the cornea is governed by the laws of refraction; specifically Snell's law, which dictates that when light impinges on a curved surface, the angle of refraction is proportional to the difference in refractive indices. The cornea, having a refractive index very close to that of water, is largely irrelevant in aquatic environments because it affects only a minor change in the angle of the incoming light rays when immersed in water regardless of the shape (Fig. 1). On the other hand, when the cornea is surrounded by air, the difference in refractive indices is increased by several orders of magnitude, turning the cornea into a refractive element that can provide most of the eye's total refractive power depending on corneal curvature. For a refractive lens, control and maintenance of curvature or shape becomes an important functional requirement for the cornea. Because of its high refractive power, even comparatively minor deviations in corneal shape introduce optical aberrations that prevent the formation of a focused image on the retina, thereby reducing visual acuity. As shape is thought to be controlled by structure, one might expect that the transition from an aquatic to a terrestrial environment associated with the evolution of fish to tetrapods (Fig. 1) may be followed by structural changes important to the regulation of corneal shape and curvature. Although some extant species of fish have evolved both terrestrial and aquatic vision (e.g., Anableps and flying fish),1,2 and tetrapods have evolved aquatic species, a comparative structural analysis of the vertebrate cornea from different vertebrate clades may help elucidate clues to the structural evolution of the cornea to a refractive lens. Figure 1 Different functional requirements for aquatic and terrestrial corneas. In a terrestrial environment (top), the difference in refractive indices between air and the cornea is large enough for the cornea to affect a significant change in direction of incoming ... We previously developed a second-harmonic generation (SHG)-based approach to three-dimensionally (3-D) reconstruct the collagen organization of the cornea at submicron resolution, called nonlinear optical high-resolution macroscopy (NLO-HRMac).3 Using HRMac, we recently began to quantify the macrostructure of the mammalian cornea, and demonstrated a link between the degree of collagen lamellar branching and corneal compliance.4 Based on these findings, we proposed that collagen lamellar branching and anastomosing, in conjunction with transverse collagen lamellar orientation respective to the corneal surface,5 stabilizes corneal biomechanics and controls corneal shape in the mammalian cornea.5,6 In this study, we tested the hypothesis that the evolution of a transparent cover to a refractive cornea involved structural changes in the collagen lamellar organization involving increased lamellar branching and anastomosis. Using HRMac, we show that corneas from cartilaginous and boney fishes have a simple orthogonally arranged, rotational lamellar organization with little lamellar branching. By comparison, higher nonmammalian vertebrates, while also maintaining an orthogonal/rotational organization, show increased lamellar branching that is greatest in birds, consistent with the hypothesis that increasing lamellar branching is associated with the evolution of a corneal refractive lens. Interestingly, the lamellar organization of the mammalian corneas is structurally unique from that of all other vertebrate clades, suggesting a previously unrecognized, evolutionary divergence.