Alfred J. Coulombre

Origins of avian ocular and periocular tissues.

The mesenchyme which surrounds the avian embryonic eye and forms all periocular skeletal and connective tissues, including the orbit and part of the cornea, is derived from two sources, the ectodermal neural crest and the mesoderm. Due to difficulties in distinguishing cells of these separate origins, the precise contributions of each to periocular tissues has not been defined. By transplanting labeled neural creast or mesoderm cells into unlabeled host embryos we have been able to catalogue first the migratory patterns of these populations and then their precise derivatives. Some donor tissues were labeled with [3H]thymidine; in other cases embryonic quail cells, which contain a replicating heterochromatin marker, were grafted into chick hosts. As the optic vesicle forms, its caudal (future temporal) surface is contacted by a sparse population of mesodermal mesenchyme; the rest of the eye is closely surrounded by superficial ectoderm. Shortly thereafter neural crest cells migrate over the caudal and rostral surfaces of the eye, forming the maxillary and frontonasal processes as well as presumptive scleral and choroidal cells. Neural crest cells form all of the skeletal and connective tissues adjacent to the medial, fnasal, ienrior, and lateral parts of the eye, including the endothelial and stromal cells of the cornea and much of the orbit. Most of the tissues behind the eye are derived from mesoderm, with the exception of the squamosal (temporal) bone. The myofibers of extrinsic ocular muscles are mesodermal, but connective tissues associated with these muscles are of neural crest origin. The ciliary muscles are formed from neural crest cells. Periocular vascular endothelial cells are all mesodermal, but peri-vascular connective tissues, including associated smooth muscles cells, are of neural crest origin. By performing heterotopic transplantations, for example replacing the chick prosencephalic neural crest with quail metencephalic crest, we have proved that neural crest cells from other regions of the head can mimic the development of periocular crest cells. This proves that the environment through which these cells migrate plays an essential role in establishing the timing and spatial patterns of their movement. Included in the environment are the basement membranes associated with epithelial tissues, such as the optic vesicle and superficial ectoderm. We have described several instances in which basement membranes underlying these epithelia become tightly apposed and appear to act as barriers to cell migration. The patterns of basement membrane apposition and subsequent separation are described and correlated with patterns of mesenchymal cell migrations.

Regeneration of neural retina from the pigmented epithelium in the chick embryo

Abstract The neural retina and lens were removed from the eyes of 4-day chick embryos, which were then divided into six groups: (a) 6 eyes were fixed immediately; (b) in 17 animals a 4-day lens alone was replaced; (c) in 32 cases both a 4-day lens and a piece of 4-day neural retina were reintroduced into the eye; (d) in 14 cases a 4-day otocyst was implanted; (e) in 13 cases a 4-day lens and a piece of 4-day optic tectum were implanted; (f) in 4 specimens a piece of 4-day neural retina alone was reintroduced into the eye. Analysis of the subsequent development of these groups led to the following observations and conclusions: 1. 1. Neural retina can regenerate from the pigmented epithelium of the chick embryo at least as late as stage 24. 2. 2. Regeneration occurs in two ways: (a) from the cup margin; (b) in patches of pigmented epithelium some distance from the margin. 3. 3. Both types of regeneration require that the neural retina be physically separated from the pigmented epithelium without being removed from the immediate environment. 4. 4. The 4-day otocyst can elicit neural retinal regeneration from the embryonic pigmented epithelium. 5. 5. The 4-day optic tectum does not elicit neural retinal regeneration from the pigmented epithelium. 6. 6. The presence of the lens is not necessary for regeneration of the neural retina. 7. 7. Regenerating neural retina differentiates more rapidly than normally developing neural retina. 8. 8. The polarity of the regenerating neural retina with respect to the eye is normal if it arises from the cup margin, and reversed if it arises in patches from the pigmented epithelium. 9. 9. The axons of the ganglion cells of the regenerating neural retina fasciculate to form a single “optic nerve” which leaves the eye at its posterior pole.

Development of the retinotectal topographic projection in the chick embryo

Abstract Known quadrants of the retina of chick embryo eyes were surgically ablated at stages before optic fibers reach the optic tectum. The resulting pattern of optic fiber distribution on the optic tectum at 12 days of incubation was determined by histological examination of serially-sectioned, silver-impregnated specimens. Partial retinal ablation at 4 or 5 days of incubation resulted at 12 days in subnormal eye size, local retinal defects and localized areas on the surface of the tectum devoid of fibers, corresponding to the quadrant of retina destroyed. In this way a map of the retinal projection upon the tectum was built up. Partial retinal ablation at 3 days resulted at 12 days in nearly normal eyes and normal optic fiber distribution on the tectum. The retinotectal connections in the chick embryo are topographically organized. The local topographic specificity of the retina becomes fixed between the third and fourth day of incubation. The demonstration that embryonic optic fibers terminate on specific local areas of the tectum indicates that the local topographic specificity of the tectum arises independently of the retina or optic fibers.

The skeleton of the eye. I. Conjunctival papillae and scleral ossicles.

Abstract The eye wall of the chicken contains a skeleton composed of membrane bone and hyaline cartilage. The membrane bones or scleral ossicles (usually 14 in number) form an imbricated ring surrounding the corneal limbus. During development, the scleral ossicles are foreshadowed by a ring of papilliform thickenings in the conjunctival epithelium. The conjunctival papillae appear during the eighth day. They correspond in number and location with the bones which subsequently develop in the mesenchyme underlying them. They begin to disappear on the twelfth day just as the ossicles are beginning to ossify. During its brief existence, a papilla undergoes a characteristic series of changes which appears to result in the liberation of PAS- and Feulgen-positive granules, as well as basement membrane-like strands into the underlying mesenchyme. If a papilla is removed surgically prior to these events the mesenchyme beneath it does not form an ossicle. Once removed the papilla does not regenerate. The number of papillae and ossicles which form depends, in part, on the rate of growth of the eye between the fourth and the eighth days. Mutual inhibition of adjacent ossicles is one of the factors that shape the ossicles.

Metaplastic induction of scales and feathers in the corneal anterior epithelium of the chick embryo

Abstract The anterior epithelium of the embryonic chick cornea has differentiated by the fourth day of incubation, and has been engaged in the deposition of a subepithelial collagenous stroma for 24 hours or longer. To test whether the corneal epithelium can be induced to participate in the formation of other ectodermal derivatives at this early stage in its differentiation, we confronted it with mesenchyme taken from feather, scale, or hair-forming regions. The lenses of 5-day chick embryos were replaced with blocks of mesenchyme derived from suitable locations in donor animals of appropriate species and age and the host eyes were examined histologically at intervals ranging up to 12 days after surgery. Dermis from the scale-forming foot region of 13-day-old donor chick embryos promoted scale formation in the anterior epithelium of most of the hosts; in some cases feathers were associated with the scales. Prospective dermis from the periotocystal feather-forming area of 5-day-old chick embryos elicited the formation of feathers in the corneal anterior epithelium of most of the hosts. Dermis from the flanks of mouse embryos at 13.5–14.5 days of gestation also produced feather formation in host corneas. The anterior epithelium of the cornea of the 5-day-old chick embryo is one of the few metazoan tissues in which a change from one relatively advanced state of differentiation to another has been induced.

Corneal development: III. The role of the thyroid in dehydration and the development of transparency

During the second half of embryonic development the cornea of the chick embryo loses water; at the same time its ability to transmit light increases. The partial dehydration of the cornea is a necessary condition for the development of corneal transparency. It occurs during the same period in development when the entire embryo is undergoing a relative dehydration. This dehydration begins as the thyroid gland becomes active, and is probably under the control of thyroxine. Exogenously supplied thyroxine, given prior to this time, accelerates both the dehydration of the cornea and its increase in transparency. On the other hand, 2-thiouracil, which inhibits the thyroid, slows down these changes. In untreated corneas, as well as in corneas treated with endocrine substances, the ability of the cornea to transmit light is an inverse linear function of its level of hydration.

Corneal development: V. Treatment of five-day-old embryos of domestic fowl with 6-diazo-5-oxo-l-norleucine (DON)☆

Abstract The embryogenesis of the corneal stroma of the domestic fowl was studied following the injection of the glutamine analog DON (6-diazo-5-oxo- l -norleucine) into the chorioallantoic veins of 5-day-old chick embryos. The 44 survivors were cilled between 1 hr and 13 days following injection. Their corneas were examined histologically and compared with those of untreated animals of similar age. All of the experimentally-treated corneas examined developed abnormally. The defects included: a temporary disappearance of portions of the endothelium; the deposition of disordered arrays of collagen fibers beneath the corneal epithelium, including a reversal in the direction of rotation of the axes of affected portions of the stromal lamellae; the appearance of stromal cysts; and the accumulation, beginning 6 days after injection, of pools of Gomori-silver-positive material within the epithelium. Abnormalities in corneal development following treatment with DON were compared with those previously obtained following administration of l -azetidine-2-carboxylic acid. The findings demonstrated that: (1) the characteristic, progressive rotation of fibril orientation which normally occurs in the outer lamellae of the avian, corneal, primary stroma is not a rigidly-determined configuration since its direction can be reversed consistently following treatment with DON; and (2) the primary stroma dictates the collagenous structure of the secondary stroma deposited within it.

The specificity of retinotectal connections studied by retinal grafts onto the optic tectum in chick embryos

Abstract Pieces of retina from known quadrants of 4-day chick embryo eyes were grafted onto the surface of the optic tectum of 6- or 7-day embryos (the contralateral eye of the hosts having been excised in an earlier operation to prevent the normal complement of optic fibers from reaching the tectum). The embryos with grafts were incubated 5 days, then prepared for histological study. Examination of serial sections stained with Protargol revealed that the retinal grafts had differentiated normally and produced fibers which passed onto the surface of the tectum as a single bundle, or as parallel fascicles. These coursed for varying distances on the surface membrane above the tectum, ending in many cases by piercing the membrane and entering the superficial tectal layers. The location of the graft on the tectal surface and the course and length of the fiber bundles were recorded, and the results grouped according to the quadrant of origin of the retinal graft. Fibers from a particular quadrant of retina preferentially grew toward specific areas of the tectum (dorsal retina to lateroventral tectum; ventral retina to dorsal tectum; nasal retina to posterodorsal tectum; temporal retina to anterodorsal tectum). These findings correlated with the known retinotopic distribution of normal optic fiber projections onto the tectum. Possible mechanisms mediating the specific guidance of fibers are discussed.

Corneal development: IV. Interruption of collagen excretion into the primary stroma of the cornea with l-azetidine-2-carboxylic acid

Abstract The primary stroma of the cornea of the chick embryo contains a cell-free orthogonal ply of collagen fibrils which is delineated clearly by Gomoris silver stain for reticulin and has, in miniature, the same fibrous architecture as the mature stroma. The collagen of this matrix is synthesized by the basal cells of the corneal epithelium and deposited beneath them a layer at a time. l -Azetidine-2-carboxylic acid (LACA; 0.5 mg in 0.005 ml of water) was injected into the chorioallantoic vein of chick embryos at 5 days of incubation, when the primary stroma is cell free. LACA is an imino acid analog of l -proline, competes with proline for incorporation into collagen and prevents the excretion of the altered collagen. Fifteen minutes after the injection of LACA the Golgi apparatuses located in the basal cytoplasm of the basal epithelial cells (which are normally silver-positive until 10 days of incubation) became silver-negative. A silver-free, collagen-sparse zone appeared beneath the corneal epithelium 1 hr after the injection of LACA. At 16 hr after LACA injection the Golgi apparatuses of the basal epithelial cells once again became silver-positive. Between 18 and 24 hr after the injection, new collagen layers began to appear once more beneath the epithelium. During the following 10 days the collagen-sparse zone became more deeply buried in the stroma and became filled with newly deposited collagen, presumably of fibrocytic origin. The corneas of control animals injected with d -azetidine-2-carboxylic acid, or simultaneously with LACA and l -proline, developed normally. The results suggest that the Golgi apparatuses of the basal cells of the corneal epithelium process the collagen which is deposited in the primary stroma, confirm previous findings that the lamellae of the primary stroma are formed in sequence just beneath the corneal epithelium and open the way for an analysis of the role of the noncollagenous stromal matrix in determining the pattern of polymerization of stromal collagen.

Cytology of the developing eye.

Publisher Summary This chapter focuses on the cytogenetic aspects of eye development. The size and curvature of the cornea; the size, shape, and position of the lens; and the location and attachments of the ciliary muscles bear well-defined spatial relationships both to each other and to the size and curvature of the retina. In addition to meeting strict geometric specifications, the cells and tissues of the eye are differentiated to serve a wide variety of functions. The outer segment of the visual cell transduces light energy into neural impulses. The tissues of the cornea, lens, vitreous substance, and retina are remarkably transparent to light and maintain refractive indices in a range suitable for image formation. The ciliary body regulates the composition and flow of aqueous humor so that an appropriate intraocular pressure is maintained. The epithelium and endothelium of the cornea regulates the water content of the stroma within the narrow limits compatible with its transparency.