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.     

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.     

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.     

Studies on the distribution of phosphorylase in eyes of the rabbit and the squirrel monkey

Summary Detailed studies on phosphorylase localization in various components of the eyeball of rabbit and squirrel monkey have been made. Corneal epithelium, endothelium, and stromal cells, extrinsic and intrinsic muscles of the eyeball, ciliary process, endothelial cells of the anterior chamber angle, vitreal cells, lens epithelium, inner segment of cones; plexiform layer, ganglion cell layer, internal and external limiting membrane and Muller cells show high phosphorylase activity. Surprisingly, we observed phosphorylase activity inside the nucleus in the posterior 2 to 3 layers of corneal epithelium. The significance of phosphorylase localization in relation to glycogen distribution in various components of the eyeball and their energy requirements is stressed.     

花背蟾蜍蝌蚪变态期角膜发育的研究

作者用光镜和电镜研究了花背蟾蜍蝌蚪变态期角膜的发育。在后肢发育晚期,内、外角膜在中央部位首先愈台,在完全变态期角膜完全愈合,此时角膜上皮细胞增殖,上皮基质变为Bowman’s膜,内、外角膜之间的成纤维细胞和由它分泌的胶原纤维形成角膜基质,内角膜细胞形成单层的角膜内皮,它与角膜基质间的Descemet’s膜最晚形成。     

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.     

Vascular response in a non-uterine site to implantation-stage embryos in the rat and guinea-pig: in vivo and ultrastructural studies

Summary Preimplantation-stage embryos were transferred to the anterior eye chamber of recipient rats and guinea-pigs. After implantation had occurred the influence of the embryo on the iris vasculature was examined ultrastructurally. In both species, the earliest effect of embryonic implantation was an increased stromal oedema. Under increasing embryonic influence the vascular endothelial cells showed an increased number of projections into the vascular lumen, while in the rat, endothelial projections were also found pushing back into the basement membrane. In the rat, the endothelium became very irregular in thickness prior to complete disintegration and loss during more advanced stages of implantation. Rat embryonic trophoblast was found invading iris vasculature, particularly in areas where the iridial endothelium was partially or completely missing. Other cells in the iris, including the stroma, appeared to be less affected. In the guinea-pig, however, trophoblast cells appeared to be capable of invading the vasculature by displacing endothelial cells that still appeared morphologically normal.     

Immunocytochemical localization of thrombomodulin in the aqueous humor passage of the rat eye

 This report describes the distribution and localization of thrombomodulin (TM) in the rat eye by light and electron microscopic immunocytochemistry. In addition to the endothelium of the entire vasculature, TM was found on the non-vascular structures lining the cavities of the posterior and anterior chambers and the limbus. TM was localized on the basal, lateral, and apical plasma membranes of the inner and outer ciliary epithelium, and the posterior iris epithelium in which there was no polarized expression of TM. In the anterior chamber, TM was localized on the luminal surface of the corneal endothelium, but was negative on the anterior border layer of the iris, which is composed of a discontinuous layer of fibroblasts and collagen fibers. Thus, TM was present at sites of cell-to-cell contact. TM was also present on the endothelia of the trabecular meshwork and the Schlemm’s canal in the limbus. TM was localized not only on the luminal plasma membrane, but also on the cytoplasmic giant vacuoles in the endothelial cells of the Schlemm’s canal. These findings extend the importance of anticoagulant mechanisms to the systems of secretion, circulation, and drainage of the aqueous humor. Accepted: 18 March 1997     

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.     

Immunohistochemical localization of S-100-containing cells in non-nervous structures of the human eye

The present study reports on the immunohistochemical distribution of S-100 antigen in non-nervous cell types within the human eye at light microscopy. In the cornea the antigen was confined to endothelial cells covering its posterior surface; the lens exhibited immunoreactivity restricted to the epithelial cells located beneath the anterior capsule. In the iris and ciliary body, S-100 was detected in stromal cells and epithelial cells of the pigmented inner layer in the former and inner epithelial cells bounding the posterior chamber in the latter.     

Potential transformation of the anterior corneal epithelium of the common frog]

The possibility of transformation of the corneal anterior epithelium in the lens following its separation from the stroma was studied. The corneal epithelium was implanted into: a) empty eye orbit and b) cavity of lensless eye of the Rana temporaria tadpoles. In the eye orbit it continued, as in the normal development, to form the basal membrane. Although in the eye cavity the structures similar to lentoids arose but the specific lens proteins were shown to be asbent from them using immunofluorescence. In both the cases, thus, no transformation of the corneal epithelium in the lens was observed. The role of stroma in the stabilization of differentiation of the corneal anterior epithelium is discussed. It is suggested that the absence of increase in the mitotic activity is one of the causes of failure of the corneal epithelium transformation in the lens.     

The keratocan gene is expressed in both ocular and non-ocular tissues during early chick development.

Extracellular matrix (ECM) keratan sulfate proteoglycans (KSPGs) are core proteins with sulfated polylactosamine side chains (KS). The KSPG core protein keratocan gene (Kera) is expressed almost exclusively in adult vertebrate cornea, but its embryonic expression is little known. Embryonic chick in situ hybridization reveals Kera mRNA expression in corneal endothelium from embryonic day (E) 4.5, Hamburger-Hamilton (HH) 25, in stromal keratocytes from E6.5, HH30, and in iris distal surface cells from E8, HH34. As highly sulfated, antibody I22-positive KS increases extracellularly from posterior to anterior across the stroma, nerves enter and populate only anterior stroma and epithelium. RT-PCR and in situ hybridization demonstrate that developmentally regulated Kera mRNA expression initiates in midbrain and dorsolateral mesenchyme at E1, HH7, then spreads caudally in hindbrain and cranial and trunk mesenchyme flanking the neural tube through E2, HH20. Cranial expression extends ventrally through the developing head, and concentrates in mesenchyme surrounding eye anterior regions and cranial ganglia, and in subepidermal pharyngeal arch mesenchyme by E3.5, HH22. Kera expression in the trunk at E3.5, HH22 and E4.5, HH25, is strong in dorsolateral subepidermal, sclerotomal and nephrogenic mesenchymes, but absent in neural tube, dorsal root ganglia, nerve outgrowths, notochord, heart and gut. Early limb buds express Kera mRNA throughout their mesenchyme, then in restricted proximal and distal mesenchymes. I22-positive KS appears only in notochord in E3.5, HH22 and E4.5, HH25, embryos. Results suggest the hypothesis that keratocan, or keratocan with minimally sulfated KS chains, may play a role in structuring ECM for early embryonic cell and neuronal migrations.     

Aquaporins in complex tissues: distribution of aquaporins 1-5 in human and rat eye

Multiple physiological fluid movements areinvolved in vision. Here we define the cellular and subcellular sitesof aquaporin (AQP) water transport proteins in human and rat eyes byimmunoblotting, high-resolution immunocytochemistry, and immunoelectronmicroscopy. AQP3 is abundant in bulbar conjunctival epithelium andglands but is only weakly present in corneal epithelium. In contrast, AQP5 is prominent in corneal epithelium and apical membranes of lacrimal acini. AQP1 is heavily expressed in scleral fibroblasts, corneal endothelium and keratocytes, and endothelium covering thetrabecular meshwork and Schlemm's canal. Although AQP1 is plentiful inciliary nonpigmented epithelium, it is not present in ciliary pigmentedepithelium. Posterior and anterior epithelium of the iris and anteriorlens epithelium also contain significant amounts of AQP1, but AQP0(major intrinsic protein of the lens) is expressed in lens fiber cells.Retinal Müller cells and astrocytes exhibit notableconcentrations of AQP4, whereas neurons and retinal pigment epitheliumdo not display aquaporin immunolabeling. These studies demonstrateselective expression of AQP1, AQP3, AQP4, and AQP5 in distinct ocularepithelia, predicting specific roles for each in the complex networkthrough which water movements occur in the 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.     

Fibronectin in the development of embryonic chick eye.

The time course of appearance and distribution of fibronectin in the developing eye have been studied in chick embryos by indirect immunofluorescence. At the 12-somite stage, fibronectin was detected as a layer under the ectodermal cells overlying the forebrain vesicle; it was also present in the head mesenchyme. During formation of the lens placode and its invagination, a zone containing fibronectin persisted around the lens as a component of the capsule. The fibronectin-containing layer was separated from the corneal epithelial cells during the formation of the acellular stroma. The migrating corneal endothelial cells were seen posterior to the fibronectin layer. The secondary stroma was strongly positive for fibronectin. Fibronectin disappeared from the cornea starting from its posterior part along with the corneal condensation. In the newborn chicken cornea, fibronectin was present only in Descemet's membrane. In addition, the embryonic vitreous body had a network of fibronectin-containing material. The distribution of fibronectin in the developing cornea, as well as other data available on this glycoprotein, is consistent with the proposed role of fibronectin in positioning and migration of cells and in organization of the extracellular matrix.