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.     

Formation of corneal endothelium is essential for anterior segment development - a transgenic mouse model of anterior segment dysgenesis

The anterior segment of the vertebrate eye is constructed by proper spatial development of cells derived from the surface ectoderm, which become corneal epithelium and lens, neuroectoderm (posterior iris and ciliary body) and cranial neural crest (corneal stroma, corneal endothelium and anterior iris). Although coordinated interactions between these different cell types are presumed to be essential for proper spatial positioning and differentiation, the requisite intercellular signals remain undefined. We have generated transgenic mice that express either transforming growth factor (alpha) (TGF(alpha)) or epidermal growth factor (EGF) in the ocular lens using the mouse (alpha)A-crystallin promoter. Expression of either growth factor alters the normal developmental fate of the innermost corneal mesenchymal cells so that these cells often fail to differentiate into corneal endothelial cells. Both sets of transgenic mice subsequently manifest multiple anterior segment defects, including attachment of the iris and lens to the cornea, a reduction in the thickness of the corneal epithelium, corneal opacity, and modest disorganization in the corneal stroma. Our data suggest that formation of a corneal endothelium during early ocular morphogenesis is required to prevent attachment of the lens and iris to the corneal stroma, therefore permitting the normal formation of the anterior segment.     

Differential expression of the HMGN family of chromatin proteins during ocular development

The HMGN proteins are a group of non-histone nuclear proteins that associate with the core nucleosome and alter the structure of the chromatin fiber. We investigated the distribution of the three best characterized HMGN family members, HMGN1, HMGN2 and HMGN3 during mouse eye development. HMGN1 protein is evenly distributed in all ocular structures of 10.5 days post-coitum (dpc) mouse embryos however, by 13.5dpc, relatively less HMGN1 is detected in the newly formed lens fiber cells compared to other cell types. In the adult, HMGN1 is detected throughout the retina and lens, although in the cornea, HMGN1 protein is predominately located in the epithelium. HMGN2 is also abundant in all ocular structures of mouse embryos, however, unlike HMGN1, intense immunolabeling is maintained in the lens fiber cells at 13.5dpc. In the adult eye, HMGN2 protein is still found in all lens nuclei while in the cornea, HMGN2 protein is mostly restricted to the epithelium. In contrast, the first detection of HMGN3 in the eye is in the presumptive corneal epithelium and lens fiber cells at 13.5dpc. In the lens, HMGN3 remained lens fiber cell preferred into adulthood. In the cornea, HMGN3 is transiently upregulated in the stroma and endothelium at birth while its expression is restricted to the corneal epithelium in adulthood. In the retina, HMGN3 upregulates around 2 weeks of age and is found at relatively high levels in the inner nuclear and ganglion cell layers of the adult retina. RT-PCR analysis determined that the predominant HMGN3 splice form found in ocular tissues is HMGN3b which lacks the chromatin unfolding domain although HMGN3a mRNA is also detected. These results demonstrate that the HMGN class of chromatin proteins has a dynamic expression pattern in the developing eye.     

OCT layered tomography of the cornea provides new insights on remodeling after photorefractive keratectomy

OCT (optical coherence tomography) of corneal layers was generated to analyze the remodeling of the epithelium and stroma after photorefractive keratectomy (PRK). Myopic PRK was performed in 15 patients. One eye underwent manual scraping of epithelium while the other was treated with Epi clear. Epi clear allowed a gentler removal of the epithelium compared to manual scraping. Scheimpflug (Pentacam, OCULUS Optikgerate Gmbh, Wetzlar, Germany) and OCT (RTVue, Optovue Inc., Fremont, California, USA) scans of the cornea were performed before and after PRK (3 months). The OCT scanner and Pentacam acquired 8 and 25 radial 2‐D scans of the cornea, respectively. The results showed similar topographic changes on the anterior corneal surface between Scheimpflug and OCT imaging. The curvature of the underlying anterior surface of the stroma after PRK was similar to the anterior corneal surface (air‐epithelium interface), when measured with OCT. Aberrometric changes were mostly similar between Scheimpflug and OCT. However, Scheimpflug imaging reported greater changes in spherical aberration and corneal higher order aberrations than OCT after PRK. This is the first study to quantify the curvatures of the stromal layers with OCT after PRK. New insights were gained, which could be useful for refinement of surgical ablation algorithms, refractive procedures and detection of ectasia.      

Corneal morphogenesis in the Indian garden lizard,Calotes versicolor (agamidae)

The present study traces corneal morphogenesis in a reptile, the lizard Calotes versicolor, from the lens placode stage (stage 24) until hatching (stage 42), and in the adult. The corneal epithelium separates from the lens placode as a double layer of peridermal and basal cells and remains bilayered throughout development and in the adult. Between stages 32– and 33+, the corneal epithelium is apposed to the lens, and limbic mesodermal cells migrate between the basement membrane of the epithelium and the lens capsule to form a monolayered corneal endothelium. Soon thereafter a matrix of amorphous ground substance and fine collagen fibrils, the presumptive stroma, is seen between the epithelium and the endothelium. Just before stage 34 a new set of limbic mesodermal cells, the keratocytes, migrate into the presumptive stroma. Migrating limbic mesodermal cells, both endothelial cells and keratocytes, use the basement membrane of the epithelium as substratum. Keratocytes may form up to six cell layers at stage 37, but in the adult stroma they form only one or two cell layers. The keratocytes sysnthesize collagen, which aggregates as fibrils and fibers organized in lamellae. The lamellae become condensed as dense collagen layers subepithelially or become compactly organized into a feltwork structure in the rest of the stroma. The basement membrane of the endothelium is always thin. Thickness of the entire cornea increases up to stage 38 and decreases thereafter until stage 41. In the adult the cornea is again nearly as thick as at stage 38.     

Dominant inhibition of lens placode formation in mice

The lens in the vertebrate eye has been shown to be critical for proper differentiation of the surrounding ocular tissues including the cornea, iris and ciliary body. In mice, previous investigators have assayed the consequences of molecular ablation of the lens. However, in these studies, lens ablation was initiated (and completed) after the cornea, retina, iris and ciliary body had initiated their differentiation programs thereby precluding analysis of the early role of the lens in fate determination of these tissues. In the present study, we have ablated the lens precursor cells of the surface ectoderm by generation of transgenic mice that express an attenuated version of diphtheria toxin (Tox176) linked to a modified Pax6 promoter that is active in the lens ectodermal precursors. In these mice, lens precursor cells fail to express Sox2, Prox1 and αA-crystallin and die before the formation of a lens placode. The Tox176 mice also showed profound alterations in the corneal differentiation program. The corneal epithelium displayed histological features of the skin, and expressed markers of skin differentiation such as Keratin 1 and 10 instead of Keratin 12, a marker of corneal epithelial differentiation. In the Tox176 mice, in the absence of the lens, extensive folding of the retina was seen. However, differentiation of the major cell types in the retina including the ganglion, amacrine, bipolar and horizontal cells was not affected. Unexpectedly, ectopic placement of the retinal pigmented epithelium was seen between the folds of the retina. Initial specification of the presumptive ciliary body and iris at the anterior margins of the retina was not altered in the Tox176 mice but their subsequent differentiation was blocked. Lacrimal and Harderian glands, which are derived from the Pax6-expressing surface ectodermal precursors, also failed to differentiate. These results suggest that, in mice, specification of the retina, ciliary body and iris occurs at the very outset of eye development and independent of the lens. In addition, our results also suggest that the lens cells of the surface ectoderm may be critical for the proper differentiation of the corneal epithelium.     

The Expression of Tenascin-X in Developing and Adult Rat and Human Eye

Tenascin-X has been studied in developing and adult rat eye and in foetal and adult human eyes, using immunohistochemistry and frozen sections. The data were compared with the distribution of tenascin-C. The immunoreactivity for tenascin-X was seen in a basement membrane-like feature in different structures of embryonic (E) day 16–17 rat eyes. Postnatal (P) day 2 and older rat eyes showed immunoreactivity for tenascin-X in different connective tissues. In the epithelial basement membrane zone of the cornea, immunostaining was positive in P5 eyes, negative in P10 and P15 eyes and again positive in P30 and adult eyes. In the 20-week-old human foetus, immunoreactivity for the tenascin was seen in the posterior parts of the conjunctival stroma adjacent to the sclera and in a basement membrane-like fashion in anterior conjunctiva. In the adult human eye, immunoreactivity for tenascin-X was seen in the anterior one-third stroma of cornea as thin fibrils, in the stroma of the limbus and conjunctiva, and in blood vessels. Immunostaining for tenascin-C was seen in the posterior aspect of the further cornea, and in mesenchyme adjacent to cornea in E16–17 rat eyes. Corneal keratocytes and Descemet's membrane showed immunoreactivity for tenascin-C in P2–P15 rat eyes. Sclera and the junction of the cornea, and sclera expressed tenascin-C in P2 and older rat eyes. In human foetal eyes, immunostaining for tenascin-C was seen in the anterior parts of the corneal stroma, in the basement membrane zone and Bowman's membrane of the corneal epithelium, in the posterior one-fifth of the corneal stroma and the sclera starting from the junction of the cornea and sclera. In normal human adult eyes, immunostaining for tenascin-X was seen in the anterior one-third stroma of cornea, in the stroma of limbus and conjunctiva, and in blood vessels. The association of tenascin-X and basement membranes in early development evokes a question of its potential function in the development of the basement membrane. The results also suggest the association of tenascin-X with connective tissue development as well as the association of tenascin-C with the migration of keratocytes during the development of the corneal stroma.     

EPIDERMAL METAPLASIA OF THE CORNEAL ANTERIOR EPITHELIUM IN THE CHICK EMBRYO

The corneal anterior epithelium of younger chick embryos can be changed into a keratinized epidermis, when it is cultured in vitro combined with 6 1/2-day dorsal dermis. Even if a Millipore filter is inserted between the corneal anterior epithelium and underlying dorsal dermis, the epithelium undergoes similar metaplastic changes. In older embryos, however, the epithelium gradually loses the competence for the keratinization. Cultivation of cornea (anterior epithelium, stroma and endothelium) of 6 1/2- or 10-day embryos results in maintenance of its original pattern, and the epithelium fails to differentiate into a keratinized epidermis. The dermis isolated from 8 1/2-day dorsal or 12 1/2-day tarsometatarsal skin is not so effective in inducing the epidermal metaplasia. The mesenchyme of 5 1/2-day proventriculus or 5 1/2-day gizzard fails to bring about any endodermal metaplasia of the corneal epithelium. The corneal stroma, on the other hand, has no inhibitory action on the keratinization of the epidermis obtained from 6 1/2-day dorsal skin.     

Direct evidence for transformation of differentiated iris epithelial cells into lens cells

Although it is generally assumed that the lens regenerated in the newt eye after complete lentectomy is formed by cells derived from the dorsal iris epithelium, experimental evidence so far obtained for this transformation does not rule out participation of cells from the dorsal iris stroma. When the normal dorsal iris epithelium of adult Notophthalmus (Triturus) viridescens was isolated and cultured in the presence of frog retinal complex, newt lens tissue was produced in 88% of cultures. These lens tissues were positive for immunofluorescence for lens-fiber-specific gamma crystallins as well as for total lens protein. On the basis of a study of stromal cells contaminating the samples of dorsal iris epithelium and a test for the lens-forming capacity in vitro of the dorsal iris stroma in the presence of frog retinal complex, it is concluded that lens formation observed in the above experiment is not dependent on the contaminating stromal cells. This implies that, in Wolffian lens regeneration, fully differentiated adult cells completely withdrawn from the cell cycle are transformed into another cell type. An additional culture experiment demonstrated that lens-forming capacity is not restricted to the dorsal half of the iris epithelium, but extends into its ventral half.     

Formation of the endothelium of the avian cornea: A study of cell movement in vivo

In the analysis of endothelial morphogenesis reported here, scanning and transmission electron microscopes and the Nomarski light microscope were used to study both untreated and manipulated eyes of chick embryos. We found that migration of the cells into the corneal area is preceded at stage 22 by a movement of macrophages between the lens and posterior surface of the corneal stroma. At stage 23, endothelial cells move out mainly from the nasal and temporal edges of the eye where they were associated with vascular (primary) mesenchyme. Initially, they migrate through a fibrous matrix which occupies the space between lens and optic lip. When the endothelial cells reach the stroma and capsule of the lens, they can use both these surfaces as substrata, even though they seem to be more adherent to the stroma. By stage 25, the endothelium is complete and covered with fibrous matrix, which now fills and may help form the anterior chamber. The cells, initially mesenchymal, now differentiate to become epithelial (a characteristic of primary mesenchyme). The migrating endothelial cells have extended lamellipodia and filopodia along their leading edges; they show no evidence of ruffling. Moreover, contact inhibition alone does not cause them to monolayer; the presence of the lens is essential to prevent multilayering of the newly formed endothelium. In the discussion, the role of extracellular matrix and tissue boundaries in directing cell migration in vivo is emphasized.     

GROWTH OF LENS AND OCULAR ENVIRONMENT: ROLE OF NEURAL RETINA IN THE GROWTH OF MOUSE LENS AS REVEALED BY AN IMPLANTATION EXPERIMENT

The lens of 6-day-old normal mouse was implanted into the lentectomized eye of adult mouse to examine the effect of retina upon the growth of the implanted lens in vivo. The implanted lens grew normally and its transparency was kept for more than 5 months after implantation. The connection between the implanted lens and the ciliary part of the recipient iris was well established with the regeneration of zonular fibers from the recipient. In young lenses implanted reversely into adult eyes, the epithelial cells facing the retina elongated and a new epithelium was formed on the corneal side of the lens within 5 days. Young lenses implanted either in normal or reverse orientation into eyes from which the retina was previously removed did not grow. The cells of the original lens epithelium of these lenses were randomly accumulated beneath the posterior lens capsule, while the anterior portion of the implanted lenses became an epithelial structure without cell elongation. These results suggest that the growth of the implanted lens may be dependent on the retina of the adult eye.     

Studies on the nature and localization of acid phosphatase in normale and lens-regenerating urodele eyes. I. Histochemical localization

Histochemical procedures for acid phosphatase in normal and lens-regenerating eyes of the urodele Diemictylus viridescens demonstrate activity in a variety of structures. In the normal urodele eye, acid phosphatase is present in conjunctival and corneal epithelial cells and associated glands, in blood vessel endothelium and posterior epithelial cells of the iris, in the anterior lens epithelium, and in the cytoplasm of the optic nerve. Acid phosphatase in the lens-regenerating eye is localized in the same structures as in the normal eye as well as in increased amounts in the corneal epithelial cells and stromal macrophages at the lentectomy wound site and in the posterior portion of the developing lens during completion of differentiation of primary into mature lens fibers characterized by loss of many intracellular organelles. On the basis of these histochemical findings, it is proposed that hydrolytic lysosomal enzymes play an important role in the processes of cellular and intracellular destruction and synthesis which occur during Wolffian lens regeneration in the urodele.     

Fibroblast-collagen interactions in the formation of the secondary stroma of the chick cornea

Fibroblasts invade the primary corneal stroma of the 6-day-old chick embryo eye. The way in which these cells build the secondary stroma has been studied by microscope examination of the stroma during the subsequent 8 Days. Eyes were embedded in low viscosity nitrocellulose, and 30-micrometer tangential sections of cornea were cut and stained with azan (giving blue collagen and red cells). These sections were sufficiently thick to include enough cells and collagen for stromal organization to be visible under Nomarski optics. Three days after invasion, the fibroblasts extend along collagen bundles in the posterior region of the stroma; surprisingly, fibroblasts near the epithelium are more rounded. The collagen itself is organized in orthogonal bundles rather than in sheets. Measurements show that posterior bundles increase in size with time while anterior stroma si similar in diameter to primary stroma. These observations confirm that the epithelium continues to deposit primary stroma up to at least the 14th day. They show, moreover, that fibroblasts deposit collagen fibrils on extant stroma and that the farther a bundle is from the epithelium, and hence the longer the period since it was first laid down, the wider it is likely to be. Analysis of the results and existing data on hyaluronic acid levels in the stroma suggests that Bowman's membrane, the region of anterior stroma that remains uncolonized by cells, is, during this period at least, primary stroma laid down but as yet unswollen.     

Laminin alpha5-chain is expressed early and widely during the development of the anterior segment of rat eye

The distribution of a novel laminin alpha5-chain in the basement membranes of the anterior segment of rat eye was studied. Frozen sections of embryonic day (E)16--17, post-natal day (P)2, 5, 10, 15 and 30 and adult rat eyes were immunostained for laminin chains alpha2, alpha5, beta1, beta2 and gamma1 and for laminin-5, as well as for EHS-laminin, to visualize all basement membranes. Laminin alpha5-, beta1- and gamma1-chain immunoreactivities were found in the basement membranes of the inner and outer layers of optic cup, lens epithelium, further corneal epithelium and skin of the eyelids in E16--17 rat eyes. In P2 and older rat eyes, laminin alpha5-, beta1- and gamma1-chains were all seen in the basement membranes of the corneal and conjunctival epithelium, Descemet's membrane, lens epithelium, ciliary processes, blood vessels and skin of the eyelids. There was a change in the expression pattern of laminin alpha5, beta1- and gamma1-chains in Descemet's membrane from the endothelial side of the membrane (P2--P15 eyes) to both sides of the membrane after P30. Immunoreactivity for laminin-5 was weak in the basement membrane of E16--17 epidermis, but strong in the basement membrane of corneal, conjunctival and eyelid epithelium in P2 and older rat eyes. Laminin alpha2- and beta2-chains were seen in conjunctival and uveal blood vessels in P15 and older rat eyes. The laminin beta2-chain emerged into the basement membrane of conjunctival epithelium in P30 and older rat eyes, suggesting a role for the laminin beta2-chain in the maturation of conjunctiva. The results suggest that laminin alpha5-chain, possibly in laminin-10 (alpha5beta1gamma1), is early and widely expressed in the basement membranes of developing and adult rat eye and, further, that laminin alpha5-chain is a major laminin alpha-chain, partly in coexpression with the alpha3-chain of laminin-5 in the basement membranes of the anterior segment of the eye in developing and adult rats. © 1998 Chapman & Hall     

Deletion of JAM-A causes morphological defects in the corneal epithelium

Junctional adhesion molecule-A (JAM-A, JAM-1, F11R) is an Ig domain containing transmembrane protein that has been proposed to function in diverse processes including platelet activation and adhesion, leukocyte transmigration, angiogenesis, epithelial cell shape and endothelial cell migration although its function in vivo is less well established. In the mouse eye, JAM-A protein expression is first detected at 12.5 dpc in the blood vessels of the tunica vasculosa, while it is first detected in both the corneal epithelium and lens between 13.5 and 14.5 dpc. In the corneal epithelium, JAM-A levels remain appreciable throughout life, while JAM-A immunostaining becomes stronger in the lens as the animals age. Both the cornea and lens of mice lacking an intact JAM-A gene are transparent until at least a year of age, although the cells of the JAM-A null corneal epithelium are irregularly shaped. In wild-type mice, JAM-A protein is found at the leading edge of repairing corneal epithelial wounds, however, corneal epithelial wound repair was qualitatively normal in JAM-A null animals. In summary, JAM-A is expressed in the corneal epithelium where it appears to regulate cell shape.     

The roles of Pax6 in the cornea,retina, and olfactory epithelium of the developing mouse embryo

The roles of Pax6 were investigated in the murine eye and the olfactory epithelium by analysing gene expression and distribution of Pax6(-/-) cells in Pax6(+/+) <--> Pax6(-/-) chimeras. It was found that between embryonic days E10.5 and E16.5 Pax6 is autonomously required for cells to contribute fully not only to the corneal epithelium, where Pax6 is expressed at high levels, but also to the to the corneal stroma and endothelium, where the protein is detected at very low levels. Pax6(-/-) cells contributed only poorly to the neural retina, forming small clumps of cells that were normally restricted to the ganglion cell layer at E16.5. Pax6(-/-) cells in the retinal pigment epithelium could express Trp2, a component of the pigmentation pathway, at E14.5 and a small number went on to differentiate and produce pigment at E16.5. The segregation and near-exclusion of mutant cells from the nasal epithelium mirrored the behaviour of mutant cells in other developmental contexts, particularly the lens, suggesting that common primary defects may be responsible for diverse Pax6-related phenotypes.     

Metaplastic transformation of the tissue of the eye in tadpoles and adult Xenopus laevis frogs]

The pigment epithelium of the tadpoles and adults X. laevis, as well as of other anurans and cyprinids, is not capable of transformation into the retina without the special influences of agents produced by the retina. When implanting a layer of pigmented epithelium of tadpoles with the Bruch's membrane into the cavity of lensless eye of a tadpole, the transformation of pigment epithelium into retina proceeded in 40% of cases and when implanting the pigment epithelium of adults without the Bruch's membrane, the transformation proceeded in 68% of cases. The lens regeneration from the cornea which proceeds simultaneously under the retina influence exerted no effect upon the metaplasia of pigmented epithelium.