Morphogenesis of the anterior segment in the zebrafish eye

Background  

The ocular anterior segment is critical for focusing incoming light onto the neural retina and for regulating intraocular pressure. It is comprised of the cornea, lens, iris, ciliary body, and highly specialized tissue at the iridocorneal angle. During development, cells from diverse embryonic lineages interact to form the anterior segment. Abnormal migration, proliferation, differentiation, or survival of these cells contribute to diseases of the anterior segment such as corneal dystrophy, lens cataract, and glaucoma. Zebrafish represent a powerful model organism for investigating the genetics and cell biology of development and disease. To lay the foundation for genetic studies of anterior segment development, we have described the morphogenesis of this structure in zebrafish.     

Automatic identification of anterior segment eye abnormality

The eyes are complex sensory organs and are designed to optimize vision under conditions of varying light. There are a number of eye disorders that can influence vision. Eye disorders among the elderly are a major health problem. With advancing age, the normal function of eye tissues decreases and there is an increased incidence of ocular pathology. The most common symptoms elicited from ocular diseases are few in number and non-specific in nature: blurred vision, pain, and redness. Cataracts occur most frequently in older people and have significant impact on an individual's quality of life. There are effective therapies and visual aids for these potential vision-limiting conditions. Corneal haze a complication of refractive surgery is characterized by the cloudiness of the normally clear cornea. Iridocyclitis is the inflammation of the Iris and ciliary body. In corneal arcus are white circles in the cornea of the eye caused by fatty deposits. So, there is a need to diagnose to the normal eye from the abnormal one. This paper presents an identification of normal eye image and abnormal (consists of five kinds of eye images) classes using radial basis function (RBF) classifier. The features are extracted from the raw images using the image processing techniques and fuzzy K-means algorithm. Our system uses 150 subjects, consisting of five different kinds of eye disease conditions. We demonstrated a sensitivity of 90%, for the classifier with the specificity of 100%. Our systems are ready clinically to run on large amount of data sets.     

Substance P-immunoreactive nerve fibres in the anterior segment of the rabbit eye

Summary Substance P-immunoreactive nerve terminals were found in several locations in the anterior segment of the rabbit eye. In the iris they occurred in the sphincter muscle and were randomly distributed in the iris stroma with some fibres running close to the dilator muscle. In the ciliary body these immunoreactive elements were few and occurred within bundles of nerve fibres, while in the ciliary processes they were more numerous with a predominantly subepithelial location. Blood vessels in the anterior uvea were often surrounded by substance P-immunoreactive fibres. No substance P-fibres were found in the cornea, while the sclera contained very few such elements.Using conventional in vitro techniques it was found that the sphincter pupillae muscle of the iris responded to electrical stimulation with a contraction that was resistant to cholinergic and adrenergic blockade, but was inhibited by the neuronal blocker tetrodotoxin. This indicates the existence of a non-cholinergic, non-adrenergic neuronal mediator of the contractile response. Exogenously applied substance P produced a long-lasting contraction of the spincter muscle, an observation compatible with the view that substance P is the noncholinergic, non-adrenergic neurotransmitter involved.     

Gonadotropin-releasing hormone receptor binding in bovine anterior pituitary

Synthetic gonadotropin-releasing hormone (GnRH) was monoiodinated at a high specific radioactivity with 125I. The iodinated hormone retained full biological activity as assessed by the release of luteinizing hormone in vitro from bovine anterior pituitary tissue slices. Specific binding of 125I-labeled gonadotropin-releasing hormone of high affinity and low capacity was obtained using dispersed bovine anterior pituitary cells. The binding had sigmoid characteristics, compatible with the presence of more than one binding site. The subcellular fraction responsible for binding was identified with the plasma membranes. However, significant binding also occurred in the secretory granules fraction. The plasma membranes were solubilized with sodium dodecyl sulfate. Using gonadotropin-releasing hormone covalently coupled to a solid phase, a protein was purified by an affinity technique from the solubilized plasma membrane preparation which possessed similar binding propperties as plasma membranes, both intact and solubilized. The protein migrated as a single component on polyacrylamide gel in sodium dodecyl sulfate and the estimated molecular weight was 60 000. The character of the gonadotropin-releasing hormone concentration dependence binding as well as association kinetics were multiphasic and suggested the presence of more than one binding site. When analyzed by the Hill plot, the Hill coefficient of all binding curves was always greater than one which is compatible with positive cooperativity. This was further supported by the dissociation studies where the dissociation rate was inversely proportionate to both the gonadotropin-releasing hormone concentration and the time interval during which the gonadotropin-releasing hormone-gonadotropin-releasing hormone receptor protein complex was formed. Using difference chromatography, aggregation of the purified gonadotropin-releasing hormone receptor protein was demonstrated to occur upon its exposure to gonadotropin-releasing hormone. The formed macromolecular complexes bound preferentially 125I-labeled gonadotropin-releasing hormone. It is concluded that a single receptor protein is responsible for gonadotropin-releasing hormone binding in the bovine anterior pituitary. It is a part of the plasma membranes. Its interaction with gonadotropin-releasing hormone provokes transitions of the protein into different allosteric forms and this may be related to the biological effect of gonadotropin-releasing hormone on gonadotropin secretion.     

Lectin binding and enzymatic deglycosylation of the cGMP-gated channel from bovine rod photoreceptors

In order to investigate the lectin-binding properties of the photoreceptor cGMP-gated channel, solubilized and purified channel protein was incubated with immobilized lectins followed by reconstitution of unbound proteins. Of the lectins tested, only concanavalin A (ConA) was able to specifically sediment channel activity. A 240-kDa protein, which copurifies with the 63-kDa channel protein but does not bind ConA, was also found to be sedimented by the ConA-affinity matrix, thereby implicating that it is associated with the channel complex. Treatment of the purified channel protein with the enzyme glycopeptidase F in the presence of the denaturing detergent sodium dodecyl sulfate resulted in a rapid reduction of the apparent molecular mass by 1.90 kDa, and the abolition of ConA-binding. No intermediate molecular weight species were observed, suggesting that the channel protein is N-glycosylated at one site only. Under nondenaturing conditions, the kinetics of deglycosylation were distinctly two-phased: 50-60% deglycosylation was achieved rapidly; however, prolonged incubation was required to arrive at complete deglycosylation. Reconstitution experiments showed that deglycosylation had no significant effect on the kinetics of channel protein activation by cGMP.     

A comparative study of the adrenergic nerves to the anterior eye segment of some primates

Summary The distribution of adrenergic terminals to the anterior eye segment of humans, Cynomolgue monkeys, squirrel monkeys, owl monkeys, Cebus monkeys, vervets, tamarins, and baboons has been investigated. The cornea is normally devoid of adrenergic terminals, except in a plexus near the limbus. The trabecular meshwork contains varying numbers of adrenergic terminals: usually none in Cynomolgus monkeys, patas monkeys, vervets, and humans, although fibres have very rarely been observed in Cynomolgus monkeys, vervets, and humans; a few in owl monkeys, squirrel monkeys, and tamarins; and moderate numbers in Cebus monkeys and baboons. From the evidence, however, it seems premature to presume an adrenergic innervation of the trabecular mechanism regulating the outflow resistance. The dilatator pupillae is regularly supplied with numerous adrenergic terminals and in the iris stroma there is probably an adrenergic innervation of the melanophores. The sphincter pupillae regularly contains adrenergic terminals with notable species differences; most fibres occur in baboons and fewest in humans, with the remaining species forming a middle class. The ciliary processes in all species contain a moderate number of adrenergic terminals, presumably primarily associated with the epithelium. Intraepithelial adrenergic terminals have been observed on the pars plana of the ciliary body of humans, Cebus monkeys, vervets, baboons, and patas monkeys. The ciliary muscle of baboons and Cynomolgus monkeys contains numerous adrenergic terminals. Moderate numbers occur in Cebus monkeys and vervets, and still less in (in falling order) tamarins, squirrel monkeys, humans, and patas monkeys.     

Lectin binding patterns in two cultured endothelial cell types derived from bovine corpus luteum

Epithelial cells of different phenotypes derived from bovine corpus luteum have been studied intensively in our laboratory. In this study, specific lectin binding was examined for cells of type 1 and 3, which were defined as endothelial cells. In order to confirm differences in their glycocalyx at the light microscopic level, five biotinylated lectins were applied to postconfluent cultures which had been fixed with buffered paraformaldehyde or glutaraldehyde. Cells were not permeabilized with any detergent. Lectin binding was localized with a streptavidin-peroxidase complex which was visualized with two different techniques. The DAB technique detected peroxidase histochemically, while the immunogold technique used an anti-peroxidase gold complex together with silver amplification. Neither cell type 1 nor cell type 3 bound a particular lectin selectively, yet each cell type expressed a particular lectin binding pattern. With the DAB technique, diverse lectin binding patterns were seen, probably indicating either outside binding, i.e., a diffuse pattern, a lateral-cell-side pattern and a microvillus-like pattern, or inside binding, i.e., a diffuse pattern, and a granule-like pattern. With the immunogold technique, only outside binding was observed. In addition, the patterns of single cilia or of single circles were detected, the latter roughly representing 3-m-sized binding sites for concanavalin A. When localizing them at the ultrastructural level, single circles corresponded with micron-sized discontinuities of the plasma membrane. Shedding vesicles were detected whose outer embrane was labelled with concanavalin A. Our results confirm the diversity of the two cell types under study. The inside lectin binding may be caused by way of transient plasma membrane openings and related to shedding of right-side out vesicles (ectocytosis).     

Cellular retinol binding protein 1 modulates photoreceptor outer segment folding in the isolated eye

In a previous study, we used differential proteomics to identify retinal proteins whose steady‐state levels were altered in an experimental system in which photoreceptor outer segments were improperly folded. We determined that the steady‐state level of cellular retinol binding protein 1 (CRBP1) was downregulated in eyes lacking organized outer segments. The purpose of this study was to determine if CRBP1 is a plausible candidate for regulating outer segment assembly. We used Morpholinos to directly test the hypothesis that a decreased level of CRBP1 protein was associated with the misfolding of outer segments. Results from these studies indicate that downregulation of CRBP1 protein resulted in aberrant assembly of outer segments. Because CRBP1 plays a dual role in the retina—retinal recycling and generation of retinoic acid—we evaluated both possibilities. Our data demonstrate that outer segment folding was not modified by 11‐cis retinal supplementation, suggesting that CRBP1 influences outer segment assembly through a mechanism unrelated to rhodopsin regeneration. In contrast, retinoic acid is required for the proper organization of nascent outer segment membranes. The localization of CRBP1 within Muller cells and the RPE and its demonstrated role in modulating the proper folding of nascent outer segment membranes through retinoic acid further elucidates the role of these cells in directly influencing photoreceptor physiology. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 623–635, 2010     

Lectin binding in the diabetic rat kidney

In this study metal-conjugated concanavalin A (Con A) and Bandieraea simplicifolia isolectin II (BSA II) have been applied to sections from kidneys of control rats and rats which had untreated diabetes for 70 days or for 200 days. Lectin binding was measured by atomic absorption spectrophotometric analysis of ferritin-iron or hemocyanin-copper. Con A binding increased significantly with diabetes; was totally blocked by alpha-D-mannoside; was not inhibited by fructose lysine; and was enhanced by NaHB4 preincubation. BSA II binding also increased significantly with diabetes.     

Lectin binding in the diabetic rat kidney

Summary In this study metal-conjugated concanavalin A (Con A) andBandieraea simplicifolia isolectin II (BSA II) have been applied to sections from kidneys of controls rats and rats which had untreated diabetes for 70 days or for 200 days. Lectin binding was measured by atomic absorption spectrophotometric analysis of ferritin-iron or hemocyanin-copper.Con A binding increased significantly with diabetes; was totally blocked by alpha-D-mannoside; was not inhibited by fructose lysine; and was enhanced by NaHB₄ preincubation. BSA II binding also increased significantly with diabetes.     

Lectin binding in the human foetal testis

In the present study we have investigated the oligosaccharidic content of the glycoconjugates within the human foetal testis starting from its earliest differentiation phase (8, 10 and 12 weeks of gestation). To this purpose we have used a battery of six horseradish peroxidase-labelled lectins (SBA, PNA, WGA, UEAI, LTA and ConA). We have obtained a complete distributional map of the sugar residues of the glycoconjugates in the coelomic mesothelium, tunica albuginea, pre-Sertoli cells, pre-gonocytes, Leydig cells, basement membrane of the sex cords, interstitial tissue, mastocytes and endothelial cells of the capillary vessels. Since the beginning of the testis differentiation phase the cells of the coelomic mesothelium showed a large amount of sugar residues. In the pre-Sertoli cells and in the pre-gonocytes a role played as structural molecules by some oligosaccharides could be hypothesized. D-galactose-(beta1-->3)-N-acetyl-D-galactosamine, sialic acid, N-acetyl-D-glucosamine and alpha-D-mannose could be involved in inducing and maintaining the cellular activity of the Leydig cells.     

The lens organizes the anterior segment: specification of neural crest cell differentiation in the avian eye

During the development of the anterior segment of the eye, neural crest mesenchyme cells migrate between the lens and the corneal epithelium. These cells contribute to the structures lining the anterior chamber: the corneal endothelium and stroma, iris stroma, and trabecular meshwork. In the present study, removal of the lens or replacement of the lens with a cellulose bead led to the formation a disorganized aggregate of mesenchymal cells beneath the corneal epithelium. No recognizable corneal endothelium, corneal stroma, iris stroma, or anterior chamber was found in these eyes. When the lens was replaced immediately after removal, a disorganized mass of mesenchymal cells again formed beneath the corneal epithelium. However, 2 days after surgery, the corneal endothelium and the anterior chamber formed adjacent to the lens. When the lens was removed and replaced such that only a portion of its anterior epithelial cells faced the cornea, mesenchyme cells adjacent to the lens epithelium differentiated into corneal endothelium. Mesenchyme cells adjacent to lens fibers did not form an endothelial layer. The cell adhesion molecule, N-cadherin, is expressed by corneal endothelial cells. When the lens was removed the mesenchyme cells that accumulated beneath the corneal epithelium did not express N-cadherin. Replacement of the lens immediately after removal led to the formation of an endothelial layer that expressed N-cadherin. Implantation of lens epithelia from older embryos showed that the lens epithelium maintained the ability to support the expression of N-cadherin and the formation of the corneal endothelium until E15. This ability was lost by E18. These studies provide evidence that N-cadherin expression and the formation of the corneal endothelium are regulated by signals from the lens. N-cadherin may be important for the mesenchymal-to-epithelial transformation that accompanies the formation of the corneal endothelium.     

The eye of the magellanic penguin (Spheniscus magellanicus): structure of the anterior segment

We undertook a light and scanning electron microscopic study of the eye in the Magellanic penguin (Spheniscus magellanicus). The anatomical peculiarities of the eyeball shape in Sphenisciformes have been previously described by others; here, we show that they are accompanied by several modifications in the organization of the anterior segment of the eye. The main change was found in the portion of opaque sclera extending from the cornea to the anterior border of the scleral ossicles, which was much broader than in other avian eyes. This scleral region was made of a very dense fibrous tissue and was as difficult to cut as the ossicles. The corneo-scleral boundary was also different from that of other birds, since the aqueous humor channel and the pectinate ligament were located 1.0-1.5 mm posterior to the cornea. The osseous ring was formed by 13 bones, including three pairs of over- and underplates. There was a single ciliary muscle, with meridionally oriented striated fibers. They were inserted on a circumference along the boundary between the fibrous sclera and the ossicles, far away from the wall of the aqueous humor channel. On their posterior end, the muscle fibers formed a tendinous structure attached to the inner surface of the sclera and to the outer surface of the ciliary body. Only short zonular fibrils were observed. These anatomical features are probably relevant for the adaptation of penguin eyes to vision on land and in the aquatic environment.     

Lectin binding by eosinophils

Lectins which identify carbohydrates and glycoproteins have been used to characterize specific components of the surface of guinea pig peritoneal exudate eosinophils. Agglutination of eosinophils purified by discontinuous metrizamide gradients was scored microscopically. Wheat germ agglutinin (WGA) was most effective (0.05 micrograms/ml). However, higher concentrations of soybean lectin and concanavalin A (Con A) were also effective. No differences in lectin binding were noted between eosinophils harvested from uninfected animals, Trichinella spiralis-infected animals, or animals receiving weekly intraperitoneal injections of polymyxin B. Neuraminidase pretreatment to remove surface sialic acid reduced agglutination by WGA. Eosinophils did not adhere to WGA-coated Sepharose beads; however, they did adhere to Con A-coated beads. Pretreatment with neuraminidase did not affect the adherence of eosinophils to plastic surfaces, suggesting that sialic acid does not play an important role in adherence. Formation of lectin-inhibitor complexes within the incubation mixture complicated interpretation of studies of binding to plastic surfaces. These studies demonstrate that lectin binding sites are present on the surface of eosinophils. Lectin-type binding may be important in interactions between eosinophils and noningestible parasites.