Chick Cornea Research Articles

Expression and Localization of Neural Cell Adhesion Molecule and Polysialic Acid during Chick Corneal Development

To assay for expression and localization of neural cell adhesion molecule (NCAM) and polysialic acid (polySia) in the chick cornea during embryonic and postnatal development. Real time quantitative PCR and Western blot analyses were used to determine NCAM expression and polysiaylation in embryonic, hatchling, and adult chick corneas. Immunofluorescence staining for NCAM and polySia was conducted on cryosections of embryonic and adult corneas, whole embryonic corneas, and trigeminal neurons. NCAM and ST8SiaII mRNA transcripts peaked by embryonic day (E)9, remained steady between E10 and E14 and slowly decreased thereafter during embryonic development. Both gene transcripts showed > 190-fold decline in the adult chick cornea compared with E9. In contrast, ST8SiaIV expression gradually decreased 26.5-fold from E6 to E19, increased thereafter, and rose to the early embryonic level in the adult cornea. Western blot analysis revealed NCAM was polysialylated and its expression developmentally changed. Other polysiaylated proteins aside from NCAM were also detected by Western blot analysis. Five NCAM isoforms including NCAM-120, NCAM-180 and three soluble NCAM isoforms with low molecular weights (87-96 kDa) were present in chick corneas, with NCAM-120 being the predominate isoform. NCAM was localized to the epithelium, stroma, and stromal extracellular matrix (ECM) of the embryonic cornea. In stroma, NCAM expression shifted from anterior to posterior stroma during embryonic development and eventually became undetectable in 20-week-old adult cornea. Additionally, both NCAM and polySia were detected on embryonic corneal and pericorneal nerves. NCAM and polySia are expressed and developmentally regulated in chick corneas. Both membrane-associated and soluble NCAM isoforms are expressed in chick corneas. The distributions of NCAM and polySia in cornea and on corneal nerves suggest their potential functions in corneal innervation.

Alterations in the Biomolecular Signatures of Developing Chick Corneas as Determined by Biospectroscopy and Multivariate Analysis

Biospectroscopy tools are increasingly being recognized as novel approaches toward interrogating complex biological structures in a nondestructive fashion. This study was conducted to apply these tools to interrogate alterations in the molecular signatures of developing chick corneas during the onset and development of transparency. Embryonic chick corneas (n = 46) were obtained at 2-day intervals from embryonic day (E)10 to E18 of incubation and investigated with attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy and Raman microspectroscopy. Resultant spectra were analyzed for variance by using principal component analysis and linear discriminant analysis (PCA-LDA). Mean spectra after ATR-FTIR spectroscopy or Raman microspectroscopy derived from corneas at each developmental stage showed some overlap; however, in PCA-LDA scores plots, a clear segregation of spectra was evident, and two-category discrimination indicated that significant molecular alterations occur during tissue morphogenesis. Notable by both techniques was the increasing intensity of DNA signal (1080 cm⁻¹) from E10 onward. Major segregating biomarkers identified by ATR-FTIR spectroscopy between E10 and E18 were in the DNA/RNA (1126 cm⁻¹), glycogen (1045 cm⁻¹), protein (1470 cm⁻¹), and amide II (1512 cm⁻¹ and 1524 cm⁻¹) spectral regions. Raman spectroscopy also identified major distinguishing vibrational modes that included proteins, amino acids (tyrosine, proline phenylalanine, and valine), and secondary structures of proteins (amide I and amide II). The developing chick cornea undergoes significant changes in its biomolecular composition in the E10 to E18 developmental period, with the major changes occurring in the spectral regions associated with DNA/RNA, proteins, glycogen, and secondary protein structures.

New Strategy to Study Corneal Endothelial Cell Transplantation: The Chick Cornea Model

To set up a culture assay of chick corneal endothelial cells (CECs) for transplantation into host corneas. Histology sections were performed at 6, 9, 12, 15, and 18 embryonic days of development of the chick embryo. Visualization of the gross morphology of CECs and of epithelium, stroma, and Descemet membrane was performed. Transplantation of CECs at 18 embryonic days of development into explanted, denuded from endothelial cell, host corneas of the same stage was attempted. The results from the histological sections clearly indicate that after embryonic day 12, the endothelial cells are well differentiated and the proliferation is complete. Transplanted CECs were able to migrate and integrate into the denuded host corneas. This study demonstrated its feasibility using an easy accessible model of chick cornea. With this technique, sufficient CECs may be obtained for biochemical and functional investigations using only nonhatched chickens that are easily accessible and easy to manipulate.

Femtosecond infrared intrastromal ablation and backscattering-mode adaptive-optics multiphoton microscopy in chicken corneas

The performance of femtosecond (fs) laser intrastromal ablation was evaluated with backscattering-mode adaptive-optics multiphoton microscopy in ex vivo chicken corneas. The pulse energy of the fs source used for ablation was set to generate two different ablation patterns within the corneal stroma at a certain depth. Intrastromal patterns were imaged with a custom adaptive-optics multiphoton microscope to determine the accuracy of the procedure and verify the outcomes. This study demonstrates the potential of using fs pulses as surgical and monitoring techniques to systematically investigate intratissue ablation. Further refinement of the experimental system by combining both functions into a single fs laser system would be the basis to establish new techniques capable of monitoring corneal surgery without labeling in real-time. Since the backscattering configuration has also been optimized, future in vivo implementations would also be of interest in clinical environments involving corneal ablation procedures.

The effects of light regimes and hormones on corneal growth in vivo and in organ culture

When chicks are exposed to constant light (CL) during growth, their corneas become flatter and lighter in weight, and their anterior segments become shallower than those of chicks exposed to cyclical periods of light and dark. These effects have been correlated with CL suppression of cyclical changes in melatonin levels. The question of whether light directly influences corneal growth (e.g. via cryptochromes in the cornea) or acts remotely via the suppression of the melatonin rhythm has not yet been answered. Retinoic acid (RA), an ubiquitous morphogen, also causes non-functional flattening during corneal growth, but its effect in vivo has not been correlated with light regimes. We wished to characterize and distinguish between hormonal and light effects on corneal growth. We used organ culture to study the direct effects of light regimes, melatonin, and RA, and compared these results with those of parallel in vivo experiments. In this study, eye drops containing melatonin or RA were applied to corneas exposed to CL in vivo or in organ culture, and effects on corneal mass and hydration were measured. We applied a melatonin blocker, luzindole, to chick corneas in normal light/dark conditions to confirm that the observed melatonin effects are mediated at the cell membrane. Anterior chamber depth and refraction in vivo were measured. We found that, during CL exposure, combined application of melatonin and RA eye drops increased the depth of the anterior segment in vivo, (P = 0.003) and interestingly, both also reduced the hyperopia of CL exposure after 2 weeks (P = 0.002), thus partially reversing the effects of CL. RA increased corneal hydration in vivo (P = 0.030) but not in organ culture. Melatonin had no effect on corneal hydration in vivo, but in organ culture, melatonin significantly decreased hydration (P < 0.001). We found no evidence for a direct effect of light on corneal hydration in growing chick corneas in culture. Melatonin is required for normal corneal growth in vivo, and together melatonin and RA, or RA alone, affects the regulation of water content within the chick cornea. Melatonin also affects corneal hydration in vitro, but RA does not.

Analysis of Corneal Stroma Organization With Wavefront Optimized Nonlinear Microscopy

To investigate the organization of stromal collagen in healthy ex vivo corneas of different species from second harmonic generation (SHG) microscopy images. A custom backscattered nonlinear microscope has been used to study the corneal structure of different species: porcine, bovine, rabbit, rat, chicken, and humans. The instrument uses a femtosecond laser for illumination, a scanning unit, and a photon-counting detection device. It also includes a wavefront aberration control module. SHG signals produced by collagen within the cornea were acquired. A motorized stage allowed optical sectioning across the entire corneal thickness. Samples were neither fixed nor stained, and they were fully scanned. SHG images revealed the microscopic organization of the lamellae of collagen fibers. Despite absorption, for all corneal depths, images could be analyzed. The anterior stroma was similar in all samples, showing interwoven short bands of collagen randomly distributed. The lamellae at the central and posterior stroma were densely packed and often presented longer bundles lying predominantly parallel to the corneal surface with characteristic spatial distributions for each species. In particular, collagen bundles in bovine and porcine corneas were interweaved. In the chick cornea, the stromal arrangement had an orientation changing regularly with depth. In human corneas, lamellae were longer and had similar orientation than their neighbors. Using a unique wavefront aberration-controlled backscattered nonlinear microscope, changes in corneal morphology as a function of depth were characterized for different species (including humans). This allowed a direct comparison among species, which might help to establish the basis of collagen distribution in animal models or to understand diseased corneas.

Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy

In this protocol, we describe a 3D imaging technique known as 'volume electron microscopy' or 'focused ion beam scanning electron microscopy (FIB/SEM)' applied to biological tissues. A scanning electron microscope equipped with a focused gallium ion beam, used to sequentially mill away the sample surface, and a backscattered electron (BSE) detector, used to image the milled surfaces, generates a large series of images that can be combined into a 3D rendered image of stained and embedded biological tissue. Structural information over volumes of tens of thousands of cubic micrometers is possible, revealing complex microanatomy with subcellular resolution. Methods are presented for tissue processing, for the enhancement of contrast with osmium tetroxide/potassium ferricyanide, for BSE imaging, for the preparation and platinum deposition over a selected site in the embedded tissue block, and for sequential data collection with ion beam milling; all this takes approximately 90 h. The imaging conditions, procedures for alternate milling and data acquisition and techniques for processing and partitioning the 3D data set are also described; these processes take approxiamtely 30 h. The protocol is illustrated by application to developing chick cornea, in which cells organize collagen fibril bundles into complex, multilamellar structures essential for transparency in the mature connective tissue matrix. The techniques described could have wide application in a range of fields, including pathology, developmental biology, microstructural anatomy and regenerative medicine.

In vivo measurement of central corneal thickness in normal chicks (Gallus gallus domesticus) with the optical low-coherence reflectometer

To determine the practicability and accuracy of central corneal thickness (CCT) measurements in living chicks utilizing a noncontact, high-speed optical low-coherence reflectometer (OLCR) mounted on a slit lamp. Twelve male chicks (Gallus gallus domesticus). Procedures Measurements of CCT were obtained in triplicate in 24 eyes of twelve 1-day-old anaesthetized chicks using OLCR. Every single measurement taken by OLCR consisted of the average result of 20 scans obtained within seconds. Additionally, corneal thickness was determined histologically after immersion fixation in Karnovsky's solution alone (20 eyes) or with a previous injection of the fixative into the anterior chamber before enucleation (4 eyes). Central corneal thickness measurements using OLCR in 1-day-old living chicks provide a rapid and feasible examination technique. Mean CCT measured with OLCR (189.7 ± 3.34 μm) was significantly lower than histological measurements (242.1 ± 47.27 μm) in eyes with fixation in Karnovsky's solution (P = 0.0005). In eyes with additional injection of Karnovsky's fixative into the anterior chamber, mean histologically determined CCT was 195.2 ± 8.25 μm vs. 191.9 ± 8.90 μm with OLCR. A trend for a lower variance was found compared to the eyes that had only been immersion fixed. Optical low-coherence reflectometry is an accurate examination technique to measure in vivo CCT in the eye of newborn chicks. The knowledge of the thickness of the chick cornea and the ability to obtain noninvasive, noncontact measurements of CCT in the living animal may be of interest for research and development of eye diseases in chick models.

The influence of lamellar orientation on corneal material behavior: biomechanical and structural changes in an avian corneal disorder.

Retinopathy, globe enlarged (RGE) is an inherited genetic disease of chickens with a corneal phenotype characterized by loss of tissue curvature and changes in peripheral collagen fibril alignment. This study aimed to characterize the material behavior of normal and RGE chicken corneas under inflation and compare this with new spatial- and depth-resolved microstructural information to investigate how stromal fibril architecture determines corneal behavior under intraocular pressure (IOP). Six RGE chicken corneas and six age-matched normal controls were tested using trephinate inflation and their stress-strain behavior determined as a function of posterior pressure. Second harmonic generation mulitphoton microscopy was used to compare the in-plane appearance and degree of through-plane interlacing of collagen lamellae between normal and mutant corneas. RGE corneas displayed a 30-130% increase in material stiffness [E(tangent)(RGE) = 0.94 ± 0.18 MPa to 3.09 ± 0.66 MPa; E(tangent)(normals) = 0.72 ± 0.13 MPa to 1.34 ± 0.35 MPa] (P ≤ 0.05). The normal in-plane disposition of anterior collagen in the peripheral cornea was altered in RGE but through-plane lamellar interlacing was unaffected. This article demonstrates changes in corneal material behavior in RGE that are qualitatively consistent with microstructural collagen alterations identified both herein and previously. This study indicates that, in general, changes in stromal fibril orientation may significantly affect corneal material behavior and thereby its response to IOP.

Ultrastructural changes in the developing chicken cornea following caffeine administration.

Caffeine is one of the most frequently consumed psychoactive substances. It has been known for many years that caffeine at high concentrations exerts harmful effects on both women's and laboratory animals' fertility, moreover it may impair normal development of many organs in the prenatal period. So far there have been few studies performed that demonstrate teratogenic effects of caffeine on structures of the developing eye, particularly the cornea. The aim of the study was to show ultrastructural changes in the developing cornea, as the effect of caffeine administration to chicken embryos. The experimental materials were 26 chicken embryos from incubated breeding eggs. Eggs were divided into two groups: control (n=30) in which Ringer liquid was administrated, and experimental (n=30) in which teratogenic dose of caffeine 3.5mg/egg was given. In 36th hour of incubation solutions were given with cannula through hole in an egg shell directly onto amniotic membrane. After closing the hole with a glass plate and paraffine, eggs were put back to incubator. In 10th and 19th day of incubation corneas were taken for morphological analysis with a use of electron microscopy. Administration of caffeine during chicken development causes changes of collagen fibers of Bowman's membrane patterns and of the corneal stroma but it also changes proportion of amount of collagen fibers and of the stromal cells.

Ultrastructural changes in the developing chicken cornea following caffeine administration.

Ultrastructural changes in the developing chicken cornea following caffeine administration.

Keratocyte‐mediated establishment of lamellar structure in corneal development

Abstract Purpose Mechanisms by which presumptive keratocytes in the developing cornea engineer the 3‐dimensional lamellar matrix of the transparent stroma remain unknown. We used Volume Scanning Electron Microscopy to observe lamella formation in embryonic avian cornea in order to understand better corneal stromal biosynthesis, transparency and repair. Methods Corneas were obtained from chick embryos at E10 and E14, when a loose structure of collagen fibril bundles condenses into the lamellar organisation of the mature stroma. Aldehyde‐fixed corneas were contrast‐enhanced in osmium ferricyanide and tannic acid and embedded in Durcupan resin. Toluidine blue‐stained sections were used to locate suitable sites, subsequently examined in unstained ultrathin sections in a Jeol 1010 transmission electron microscope. The polished faces of the resin blocks were then imaged using a BSE detector in a Quanta 3D FEG FIB/SEM via an alternate slice‐and‐view protocol. 50nm slicing was performed with a focused gallium ion beam and image series were collected over 18h. Image stacks were processed using ImageJ software. Results Transmission and volume scanning electron microscopy reveal complex compartmentalisation of developing chick corneal stroma. Keratocyte filopodia extend many microns from cells into the extracellular space. These invest collagen fibrils, with varying orientations, and persist as the stroma condenses, appearing to orchestrate the spatial and directional organisation of the component lamellae. Conclusion Volume SEM reveals in 3D the complex cellular morphology of prospective keratocytes in developing chick cornea with extensive filopodia, which presumably channel and direct collagen fibril bundles into an emergent lamellar architecture.

Changes in thickness of each layer of developing chicken cornea after administration of caffeine.

The aim of the study was the presentation of changes in thickness of each layer of a developing cornea, that came into being under an influence of caffeine which was administered to chicken embryos. Research materials were 26 chicken embryos from breeding eggs that had been incubated. Breeding eggs were divided into two groups: control (n=30) in which Ringer liquid was given, and experimental (n=30) in which teratogenic dose of caffeine was administrated - 3.5 mg/egg. In 36th hour of incubation solutions were given with cannula through a hole in an egg shell directly onto amniotic membrane. After closing the hole with paraffin, eggs were put back into incubator. On 10th and 19th day of incubation corneas were taken for morphometric and morphological analysis. In experimental groups reduction of corneal thickness, thickening of corneal epithelium and corneal endothelium as well as Bowman's and Descemet's membranes, decrease of thickness of corneal stroma in comparison with the control group have been observed. Caffeine causes thickness changes of all layers and decreases the total thickness of a developing cornea.

Temporal and spatial distribution of TGF-beta isoforms and signaling intermediates in corneal regenerative wound repair.

The present study analyzed the temporal and spatial expression of TGF-beta isoforms and activated pSmad2 and p38MAPK during epithelial debridement wound repair, using chick cornea by immunohistochemistry. Normal corneas showed low-level TGF-betas staining. Following wounding, TGF-beta1 expression was strong in the Bowman's layer (BL). TGF-beta3 expression was confined to basal cells in the regenerating and unwounded regions, and was not detected in migrating epithelial, stromal or endothelial cells. In addition, TGF-beta3 treatment stimulated the proliferation of cultured epithelial cells. Our present findings seem to suggest that the TGF-beta3 signal may be required for epithelial cell proliferation. TGF-beta2 expression was strong in migrating and proliferating epithelial cells, many active migrating fibroblasts at the wound edge, endothelial cells and Descemet's membrane (DM). Although both nuclear pSmad2 and p38MAPK staining was observed in many basal epithelial cells, pSmad2 positive cells were co-localized with PCNA positive cells. Therefore, it seems likely that the pSmad2 signal may affect epithelial cell proliferation in healing corneas. Both pSmad2 and p38MAPK expression were also observed in endothelial cells. Interestingly, many active fibroblasts over the whole stroma in early wound healing at day 2 expressed nuclear pSmad2, but little if any cytoplasmic p38MAPK. Collectively, temporal/spatial up-regulation and distribution of the three TGF-beta isoforms, as well as concerted activation of both Smad2 and p38MAPK, appears to be a key aspect of regenerative corneal wound healing in the chick.

Differential Relative Sulfation of Keratan Sulfate Glycosaminoglycan in the Chick Cornea during Embryonic Development

To investigate structural remodeling of the developing corneal stroma concomitant with changing sulfation patterns of keratan sulfate (KS) glycosaminoglycan (GAG) epitopes during embryogenesis and the onset of corneal transparency. Developing chick corneas were obtained from embryonic day (E)12 to E18 of incubation. Extracellular matrix composition and collagen fibril spacing were evaluated by synchrotron x-ray diffraction, hydroxyproline assay, ELISA (with antibodies against lesser and more highly sulfated KS), and transmission electron microscopy with specific proteoglycan staining. A significant relative increase in highly sulfated KS epitope labeling occurred with respect to hydroxyproline content in the final week of chick development, as mean collagen interfibrillar distance decreased. Small KS PG filaments increased in frequency with development and were predominantly fibril associated. The accumulation of highly sulfated KS during the E12 to E18 timeframe could serve to fine tune local matrix hydration and collagen fibril spacing during corneal growth, as gross dehydration and compaction of the stroma progress through the action of the nascent endothelial pump.

Distribution of TGF‐β isoforms and signaling intermediates in corneal fibrotic wound repair

In this study, temporal and spatial distribution of three TGF-beta isoforms and their downstream signaling pathways including pSmad2 and p38MAPK were examined during fibrotic wound repair. In normal chick corneas, TGF-beta1, -2, and -3 were weakly detected in Bowman's layer (BL). In healing corneas, TGF-beta1 was primarily deposited in the fibrin clot and the unwounded BL. TGF-beta2 was highly expressed in healing epithelial and endothelial cells, and numerous active fibroblasts/myofibroblasts. TGF-beta3 was mainly detected in the unwound region of basal epithelial cells. alpha-Smooth muscle actin (alpha-SMA) was initially appeared in the posterior region of repairing stroma at day 3, and was detected in the entire healing stroma by day 7. Notably, alpha-SMA was absent in the central region of healing stroma by day 14, and its staining pattern was similar to those of TGF-beta2 and p38MAPK. By contrast, pSmad2 was mainly detected in the fibroblasts. In normal cornea, laminin was mainly detected in both epithelial basement membrane (BM) and Descemet's membrane (DM). By contrast to reconstitution of the BM in the wound region, the DM was not repaired although endothelial layer was regenerated, indicating that high levels of TGF-beta2 were released into the posterior region of healing stroma on day 14. High levels of alpha-SMA staining, shown in cultured repair stromal cells from healing corneas on day 14 and in TGF-beta2 treated normal stromal cells, were significantly reduced by p38MAPK inhibition. Collectively, this study suggests that TGF-beta2-mediated myofibroblast transformation is mediated, at least partly, by the p38MAPK pathway in vivo.

Embryonic Corneal Schwann Cells Express Some Schwann Cell Marker mRNAs, but No Mature Schwann Cell Marker Proteins

Embryonic chick nerves encircle the cornea in pericorneal tissue until embryonic day (E)9, then penetrate the anterior corneal stroma, invade the epithelium, and branch over the corneal surface through E20. Adult corneal nerves, cut during transplantation or LASIK, never fully regenerate. Schwann cells (SCs) protect nerve fibers and augment nerve repair. This study evaluates SC differentiation in embryonic chick corneas. Fertile chicken eggs were incubated from E0 at 38 degrees C, 45% humidity. Dissected permeabilized corneas plus pericorneal tissue were immunostained for SC marker proteins. Other corneas were paraffin embedded, sectioned, and processed by in situ hybridization for corneal-, nerve-related, and SC marker gene expression. E9 to E20 corneas, dissected from pericorneal tissue, were assessed by real-time PCR (QPCR) for mRNA expression. QPCR revealed unchanging low to moderate SLIT2/ROBO and NTN/UNC5 family, BACE1, and CADM3/CADM4 expressions, but high NEO1 expression. EGR2 and POU3F1 expressions never surpassed PAX3 expression. ITGNA6/ITGNB4 expressions increased 20-fold; ITGNB1 expression was high. SC marker S100 and MBP expressions increased; MAG, GFAP, and SCMP expressions were very low. Antibodies against the MPZ, MAG, S100, and SCMP proteins immunostained along pericorneal nerves, but not along corneal nerves. In the cornea, SLIT2 and SOX10 mRNAs were expressed in anterior stroma and epithelium, whereas PAX3, S100, MBP, and MPZL1 mRNAs were expressed only in corneal epithelium. Embryonic chick corneas contain SCs, as defined by SOX10 and PAX3 transcription, which remain immature, at least in part because of stromal transcriptional and epithelial translational regulation of some SC marker gene expression.

Ultrastructural changes in the retinopathy, globe enlarged (rge) chick cornea

In the cornea, the precise organisation of fibrillar collagen and associated proteoglycans comprising the stromal extracellular matrix plays a major role in governing tissue form and function. Recently, abnormal collagen alignment was noted in the misshapen corneas of mature chickens affected by the retinopathy, globe enlarged (rge) mutation. Here we further characterize corneal ultrastructural changes as the rge eye develops post-hatch. Wide-angle X-ray scattering disclosed alteration to dominant collagen lamellae directions in the rge chick cornea, compared to age-matched controls. These changes accompanied eye globe enlargement and corneal flattening in affected birds, manifesting as a progressive loss of circumferential collagen alignment in the peripheral cornea and limbus in birds older than 1 month. Collagen intermolecular separation was unchanged in rge. However, small-angle X-ray scattering results suggest collagen fibril separation and diameter increase more rapidly towards the corneal periphery in rge at 3 months post-hatch compared to controls, although central collagen fibril diameter was unchanged. By transmission electron microscopy utilising cuprolinic blue stain, the morphology and distribution of stromal proteoglycans were unaltered in rge corneas otherwise demonstrating abnormal collagen fibril organisation. From a numerical simulation of tissue mechanics, progressive remodelling of stromal collagen in rge during globe enlargement post-hatch appears to be related to the corneal morphometric changes presented by the disease.

Actin and Type I Collagen Propeptide Distribution in the Developing Chick Cornea

To determine the organization of actin filaments and distribution of type I procollagen during the development of the chick corneal stroma. Embryonic chicken corneas of ages 6 to 18 days and 18 days posthatch were cryosectioned and fluorescently labeled for filamentous actin with phalloidin and for the N-and C-terminal propeptides of type I procollagen with specific monoclonal antibodies. Tissue sections were examined by fluorescence and confocal microscopy. Prominent actin filament bundles were present at all embryonic stages, arranged in orthogonal arrays. Type I collagen propeptides were also present, with the C-propeptide visible as small foci, often associated with the actin label. The N-propeptide was also detected in the stromal matrix, especially in Bowman's layer. Actin filaments were also prominent in the corneal epithelium, along with collagen propeptide labeling, up to embryonic day 14. Actin filament bundles are abundant in the stroma, presumably in the keratocytes of the developing chick cornea, and are arranged in an orthogonal manner suggesting a possible role in cell and matrix organization in this tissue. Filament bundles appear to be closely associated with the foci of type I procollagen label, suggesting a possible association between the actin cytoskeleton and the trafficking of collagen. The presence of the N-propeptide of type I collagen in the extracellular matrix and the restricted distribution of the C-propeptide suggest differential processing of these molecules after secretion. The persistence of the N-propeptide implies a role in development, possibly in association with control of collagen fibril diameter and spacing.

Formation of stromal collagen fibrils and proteoglycans in the developing zebrafish cornea

Collagen fibrils and proteoglycans are the main components of the corneal extracellular matrix and corneal transparency depends crucially on their proper arrangement. In the present study, we investigated the formation of collagen fibrils and proteoglycans in the developing cornea of the zebrafish, a model organism used to study vertebrate embryonic development and genetic disease. We employed thin-section electron microscopy to investigate the ultrastructure of the zebrafish cornea at different developmental stages. The layering of the zebrafish cornea into an epithelium, a Bowman's layer, stroma and endothelium was observed starting at 72 hr post-fertilization. At this stage, the stroma contained orthogonally arranged collagen fibrils and small proteoglycans. The density of proteoglycans increased gradually throughout subsequent development of the cornea. In the stroma of 2-week-old larvae, the collagen fibrils were organized into thin lamellae and were separated by very large, randomly distributed proteoglycans. At 4 weeks, a regular arrangement of proteoglycans in relation to the collagen fibrils was observed for the first time and the lamellae were also thickened. The present study, for the first time, provides ultrastructural details of collagen fibril and proteoglycan development in the zebrafish cornea. Furthermore, it directly correlates the collagen fibril and proteoglycan composition of the zebrafish cornea with that of the human cornea. The similarities between the two species suggest that the zebrafish could serve as a model for investigating the genetics of human corneal development and diseases.