Identification of a primary stroma and novel endothelial cell projections in the developing human cornea

Purpose: To investigate the initial events in the development of the human cornea, focusing on cell migration, and extracellular matrix synthesis and organization. To determine whether elastic fibers are present in the extracellular matrix during early human corneal development. Methods: Human corneas were collected from week 7 to week 17 of development. An elastic fiber-enhancing stain, tannic acid–uranyl acetate, was applied to all tissue. Three-dimensional serial block-face scanning electron microscopy combined with conventional transmission electron microscopy was used to analyze the corneal stroma. Results: An acellular collagenous primary stroma with an orthogonal arrangement of fibrils was identified in the central cornea from week 7 of corneal development. At week 7.5, mesenchymal cells migrated toward the central cornea and associated with the acellular collagenous matrix. Novel cell extensions from the endothelium were identified. Elastic fibers were found concentrated in the posterior peripheral corneal stroma from week 12 of corneal development. Conclusions: This study provides novel evidence of an acellular primary stroma in the early development of the embryonic human cornea. Cell extensions exist as part of a communication system and are hypothesized to assist in the migration of the mesenchymal cells and the development of the mature cornea. Elastic fibers identified in early corneal development may play an important role in establishing corneal shape

Three-dimensional imaging of the extracellular matrix and cell interactions in the developing prenatal mouse cornea

As the outer lens in the eye, the cornea needs to be strong and transparent. These properties are governed by the arrangement of the constituent collagen fibrils, but the mechanisms of how this develops in mammals is unknown. Using novel 3-dimensional scanning and conventional transmission electron microscopy, we investigated the developing mouse cornea, focusing on the invading cells, the extracellular matrix and the collagen types deposited at different stages. Unlike the well-studied chick, the mouse cornea had no acellular primary stroma. Collagen fibrils initially deposited at E13 from the presumptive corneal stromal cells, become organised into fibril bundles orthogonally arranged between cells. Extensive cell projections branched to adjacent stromal cells and interacted with the basal lamina and collagen fibrils. Types I, II and V collagen were expressed from E12 posterior to the surface ectoderm, and became widespread from E14. Type IX collagen localised to the corneal epithelium at E14. Type VII collagen, the main constituent of anchoring filaments, was localised posterior to the basal lamina. We conclude that the cells that develop the mouse cornea do not require a primary stroma for cell migration. The cells have an elaborate communication system which we hypothesise helps cells to align collagen fibrils

A comparative study of the elastic fibre system within the mouse and human cornea

The cornea relies on its organised extracellular matrix for maintaining transparency and biomechanical strength. Studies have identified an elastic fibre system within the human posterior cornea, thought to allow for slight deformations in response to internal pressure fluctuations within the eye. However, the type of elastic fibres that exist within the cornea and their roles remain elusive. The aim of this study was to compare the distribution and organisation of the elastic fibres within the posterior peripheral mouse and human cornea, and elucidate how these fibres integrate with the trabecular meshwork, whilst characterising the distribution of their main likely components (fibrillin-1, elastin and type VI collagen) in different parts of the cornea and adjacent sclera. We identified key differences in the elastic fibre system between the human and mouse cornea. True elastic fibres (containing elastin) were identified within the human posterior peripheral cornea. Elastic fibres appeared to present as an extensive network throughout the mouse corneal stroma, but as fibrillin-rich microfibril bundles rather than true elastic fibres. However, tropoelastin staining indicated the possibility that true elastic fibres had yet to develop in the young mice studied. Differences were also apparent within the anatomy of the trabecular meshwork. The human trabecular meshwork appeared to insert between the corneal stroma and Descemet's membrane, with elastic fibres continuing into the stroma from the trabecular meshwork anterior to Descemet's membrane. Within the mouse cornea, no clear insertion point of the trabecular meshwork was seen, instead the elastic fibres within the trabecular meshwork continued into Descemet's membrane, with the trabecular meshwork joining posterior to Descemet's membrane

Developmental abnormalities in the cornea of a mouse model for Marfan syndrome

Elastic fibres provide tissues with elasticity and flexibility. In the healthy human cornea, elastic fibres are limited to the posterior region of the peripheral stroma, but their specific functional role remains elusive. Here, we examine the physical and structural characteristics of the cornea during development in the mgΔloxPneo dominant-negative mouse model for Marfan syndrome, in which the physiological extracellular matrix of its elastic-fibre rich tissues is disrupted by the presence of a dysfunctional fibrillin-1 glycoprotein. Optical coherence tomography demonstrated a reduced corneal thickness in the mutant compared to wild type mice from embryonic day 16.5 until adulthood. X-ray scattering and electron microscopy revealed a disruption to both the elastic fibre and collagen fibril ultrastructure in the knockout mice, as well as abnormally low levels of the proteoglycan decorin. It is suggested that these alterations might be a result of increased transforming growth factor beta signalling. To conclude, this study has demonstrated corneal structure and ultrastructure to be altered when fibrillin-1 is disrupted and has provided insights into the role of fibrillin-1 in developing a functional cornea

Embryonic development of the avian sternum and its morphological adaptations for optimizing locomotion

The sternum is part of the forelimb appendicular skeleton found in most terrestrial vertebrates and has become adapted across tetrapods for distinctive modes of locomotion. We review the regulatory mechanisms underlying sternum and forelimb development and discuss the possible gene expression modulation that could be responsible for the sternal adaptations and associated reduction in the forelimb programme found in flightless birds. In three phylogenetically divergent vertebrate lineages that all undertake powered flight, a ventral extension of the sternum, named the keel, has evolved independently, most strikingly in volant birds. In flightless birds, however, the sternal keel is absent, and the sternum is flattened. We review studies in a variety of species that have analysed adaptations in sterna morphology that are related to the animal’s mode of locomotion on land, in the sky and in water

A comparative study of the mouse and human corneal elastic system [Abstract]

Purpose : To characterise and compare the elastic fibre system of mouse and human posterior cornea. Methods : 4 adult human corneas were obtained from a UK eye bank and 20 corneas taken from 3 month old C5BL/6 mice which had been euthanized. The samples were examined using Serial block face scanning electron microscopy (SBF-SEM) and transmission electron microscopy (TEM). The corneas were fixed and processed for SBF-SEM through a series of staining solutions prior to embedding and polymerization in epoxy resin blocks. The sample blocks were trimmed and transferred to a Zeiss Sigma VP FEG scanning electron microscope equipped with a Gatan 3View system, where data sets of up to 1000 images were acquired of the block surface every 50nm through automated sectioning. Selected serial image sequences were extracted and 3D reconstructions were generated using Amira 6.4 software. For electron microscopy ultrathin sections were prepared and examined in a JEOL 1010 TEM. The corneas were also examined using immunofluorescence labelling of elastin and fibrillin-1. Results : Immunofluorescence revealed an extensive fibrillin-1-rich microfibril system running throughout the mouse corneal stroma. Human corneas were also positive for fibrillin-1 within the posterior cornea, but in addition exhibited positive elastin staining confined to the posterior peripheral cornea. TEM confirmed the presence of true elastic fibres containing an amorphous elastin core in peripheral human corneas, which were absent within the mouse microfibrils. Clear structural differences at the convergence of the trabecular meshwork (TM) into the elastic fibre system were also observed between mouse and human cornea using SBF-SEM. In mouse cornea the TM merged directly with Descemet’s membrane whereas in human cornea the TM inserted into the elastic fibre system anterior to Descemet’s. Conclusions : Clear differences exist between the elastic fibre system and TM anatomy of mouse and human cornea. These differences suggest the fibre system of mouse and human have different biomechanical properties and function

A comparative study of the mouse and human corneal elastic system [Abstract]

Purpose : To characterise and compare the elastic fibre system of mouse and human posterior cornea. Methods : 4 adult human corneas were obtained from a UK eye bank and 20 corneas taken from 3 month old C5BL/6 mice which had been euthanized. The samples were examined using Serial block face scanning electron microscopy (SBF-SEM) and transmission electron microscopy (TEM). The corneas were fixed and processed for SBF-SEM through a series of staining solutions prior to embedding and polymerization in epoxy resin blocks. The sample blocks were trimmed and transferred to a Zeiss Sigma VP FEG scanning electron microscope equipped with a Gatan 3View system, where data sets of up to 1000 images were acquired of the block surface every 50nm through automated sectioning. Selected serial image sequences were extracted and 3D reconstructions were generated using Amira 6.4 software. For electron microscopy ultrathin sections were prepared and examined in a JEOL 1010 TEM. The corneas were also examined using immunofluorescence labelling of elastin and fibrillin-1. Results : Immunofluorescence revealed an extensive fibrillin-1-rich microfibril system running throughout the mouse corneal stroma. Human corneas were also positive for fibrillin-1 within the posterior cornea, but in addition exhibited positive elastin staining confined to the posterior peripheral cornea. TEM confirmed the presence of true elastic fibres containing an amorphous elastin core in peripheral human corneas, which were absent within the mouse microfibrils. Clear structural differences at the convergence of the trabecular meshwork (TM) into the elastic fibre system were also observed between mouse and human cornea using SBF-SEM. In mouse cornea the TM merged directly with Descemet’s membrane whereas in human cornea the TM inserted into the elastic fibre system anterior to Descemet’s. Conclusions : Clear differences exist between the elastic fibre system and TM anatomy of mouse and human cornea. These differences suggest the fibre system of mouse and human have different biomechanical properties and function