Three-dimensional aspects of matrix assembly by cellsin the developing cornea

Abstract

Cell-directed deposition of aligned collagen fibrils during cornealembryogenesis is poorly understood, despite the fact that it is thebasis for the formation of a corneal stroma that must be transparent to visible light and biomechanically stable. Previous studiesof the structural development of the specialized matrix in thecornea have been restricted to examinations of tissue sections byconventional light or electron microscopy. Here, we use volumescanning electron microscopy, with sequential removal of ultra-thin surface tissue sections achieved either by ablation with afocused ion beam or by serial block face diamond knife microtomy,to examine the microanatomy of the cornea in three dimensionsand in large tissue volumes. The results show that cornealkeratocytes occupy a significantly greater tissue volume thanwas previously thought, and there is a clear orthogonality incell and matrix organization, quantifiable by Fourier analysis.Three-dimensional reconstructions reveal actin-associated tubularcell protrusions, reminiscent of filopodia, but extending more than30 [im into the extracellular space. The highly extended network ofthese membrane-bound structures mirrors the alignment of collagen bundles and emergent lamellae and, we propose, playsa fundamental role in dictating the orientation of collagen inthe developing cornea.