Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 111, Issue 2, Pages 687-692Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1313561110
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Funding
- Biotechnology and Biological Sciences Resource Council [BB/f022077/1]
- Engineering and Physical Sciences Research Council (EPSRC) [EP/F034970]
- Medical Research Council [MR/K000837/1]
- EPSRC [EP/F019882/1]
- Wellcome Trust [091840/Z/10/Z, 083898/Z/07/Z, 081406/Z/06/Z]
- Wellcome Trust [091840/Z/10/Z, 083898/Z/07/Z, 081406/Z/06/Z] Funding Source: Wellcome Trust
- BBSRC [BB/F022077/1] Funding Source: UKRI
- EPSRC [EP/F019882/1, EP/F034970/1] Funding Source: UKRI
- MRC [MR/K000837/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/F022077/1] Funding Source: researchfish
- Engineering and Physical Sciences Research Council [EP/F034970/1, EP/F019882/1] Funding Source: researchfish
- Medical Research Council [MR/K000837/1] Funding Source: researchfish
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Cell-directed deposition of aligned collagen fibrils during corneal embryogenesis is poorly understood, despite the fact that it is the basis for the formation of a corneal stroma that must be transparent to visible light and biomechanically stable. Previous studies of the structural development of the specialized matrix in the cornea have been restricted to examinations of tissue sections by conventional light or electron microscopy. Here, we use volume scanning electron microscopy, with sequential removal of ultrathin surface tissue sections achieved either by ablation with a focused ion beam or by serial block face diamond knife microtomy, to examine the microanatomy of the cornea in three dimensions and in large tissue volumes. The results show that corneal keratocytes occupy a significantly greater tissue volume than was previously thought, and there is a clear orthogonality in cell and matrix organization, quantifiable by Fourier analysis. Three-dimensional reconstructions reveal actin-associated tubular cell protrusions, reminiscent of filopodia, but extending more than 30 mu m into the extracellular space. The highly extended network of these membrane-bound structures mirrors the alignment of collagen bundles and emergent lamellae and, we propose, plays a fundamental role in dictating the orientation of collagen in the developing cornea.
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