Cell-independent matrix configuration in early corneal development

Abstract

Mechanisms controlling the spatial configuration of the remarkably ordered collagen-rich extracellular matrix of the transparent cornea remain incompletely understood. We previously described the assembly of the emerging corneal matrix in the mid and late stages of embryogenesis and concluded that collagen fibril organisation was driven by cell-directed mechanisms. Here, the early stages of corneal morphogenesis were examined by serial block face scanning electron microscopy of embryonic chick corneas starting at embryonic day three (E3), followed by a Fourier transform analysis of three-dimensional datasets and theoretical considerations of factors that influence matrix formation. Eyes developing normally and eyes that had the lens surgically removed at E3 were studied. Uniformly thin collagen fibrils are deposited by surface ectoderm-derived corneal epithelium in the primary stroma of the developing chick cornea and form an acellular matrix with a striking micro-lamellar orthogonal arrangement. Fourier transform analysis supported this observation and indicated that adjacent micro-lamellae display a clockwise rotation of fibril orientation, depth-wise below the epithelium. We present a model which attempts to explain how, in the absence of cells in the primary stroma, collagen organisation might be influenced by cell-independent, intrinsic mechanisms, such as fibril axial charge derived from associated proteoglycans. On a supra-lamellar scale, fine cords of non-collagenous filamentous matrix were detected over large tissue volumes. These extend into the developing cornea from the epithelial basal lamina and appear to associate with the neural crest cells that migrate inwardly to form, first the corneal endothelium and then keratocytes which synthesise the mature, secondary corneal stroma. In a small number of experimental specimens, matrix cords were present even when periocular neural crest cell migration and corneal morphogenesis had been perturbed following removal of the lens at E3.

Observations on nascent matrix structures in embryonic cornea: Important in cell interactions, or merely vestiges of the lens surface?

“…In addition, whilst sutural fibres persist in the mature adult stoma, matrix cords have not been identified in the fully-formed avian cornea post-hatch. In our previous publication [8], we postulated that matrix cords may contribute to the development of tissue curvature, a vital property of the cornea allowing refraction of light for effective vision. This theory has yet to be tested.…”

“…Tissues examined in this report were the same as those employed previously and from which data were presented in a recent publication [8]. In brief, corneas were isolated from the eyes of white Leghorn chicken embryos between days 4 and 6 of incubation (E4-E6), providing a total of 99 specimens.…”

“…Its accessibility in the avian embryo and lesser availability in mammalian systems has meant that the majority of our knowledge of corneal development stems from observations on the chicken embryo [1][2][3][4]. Only quite recently, however, have advances in imaging techniques made it possible to observe the structure, organisation and emergence of this remarkable, regular matrix in three dimensions (3D) [5][6][7][8]. Some 3D studies on mammalian corneal development have also been reported [9,10].…”

“…Some 3D studies on mammalian corneal development have also been reported [9,10]. In particular, the development of serial block face scanning electron microscopy (SBF SEM) [11,12], which allows high resolution imaging at the level of single collagen fibrils in the x-y plane and 3D reconstruction of large tissue volumes, has provided new insights into the involvement of both cell-directed and cell-independent mechanisms in the deposition of the collagenous corneal stroma [7,8]. In our previous study using these techniques, we considered intrinsic mechanisms which might generate the orthogonal arrangement of the very first fibrils of collagen, deposited by the ectoderm as micro-lamellae in the primary stroma [8].…”

“…In particular, the development of serial block face scanning electron microscopy (SBF SEM) [11,12], which allows high resolution imaging at the level of single collagen fibrils in the x-y plane and 3D reconstruction of large tissue volumes, has provided new insights into the involvement of both cell-directed and cell-independent mechanisms in the deposition of the collagenous corneal stroma [7,8]. In our previous study using these techniques, we considered intrinsic mechanisms which might generate the orthogonal arrangement of the very first fibrils of collagen, deposited by the ectoderm as micro-lamellae in the primary stroma [8]. We also focused on 'matrix cords', string-like proximal extensions of the epithelial basement membrane and presented a model, based on engineering principles, illustrating how they might contribute to the curvature of the corneal stroma.…”