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The formation of neural retina (NR) from retinal pigmented epithelium (RPE) of chick embryos in culture was investigated. In cultures of explants of PRE, depigmented, preretinal foci, consisting of 50 to 100 cells appeared in the pigmented central portion of the explant within three days. Then these depigmented cells increased rapidly in number and by about day 14 they formed characteristic spherical bodies, which were identified as a neural retinal-like structure (NR structure) by electron microscopic observations. Culture of explants of RPE from embryos of different stages showed that the capacity of embryonic RPE to form an NR structure decreased steadily with embryonic age from st. 24 to 27. At and after stage 27, no foci leading to the neural retinal differentiation were formed in the explants. Medium conditioned by cell cultures of chicken embryonic NR, RPE or chondrocytes had no effect on the formation of NR structures by explants of RPE.

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.

The developmental period during which the lens is susceptible to one or another cataractogenic influence, the types of cataracts that result, and the spatial and temporal patterns in which these cataracts become manifest are better understood in the context of the orderly sequence of steps by which the vertebrate lens develops. Thus, events attending lens induction, formation of the lens vesicle, deposition of the lens capsule, maturation of lens fibers, elaboration of lens sutures, and developmental changes in the relationship of the lens to the optic cup have implications for cataractogenesis. We have also seen that events in juxtalenticular tissues (eg, the zonule, the temporary hyaloid-lenticular vascular, the neural retina) can contribute to the development of lenticular opacities. Hereditary cataracts, as well as congenital cataracts produced in response to untoward environmental input, can be properly understood only within a dynamic developmental context.

The primary stroma of the cornea of the chick embryo contains a cell-free orthogonal ply of collagen fibrils which is delineated clearly by Gomori's 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.

Fresh rat liver and fresh rat kidney were rapidly minced and homogenized. Repeated refrigerated centrifugations of each homogenate, each time at higher speeds, yielded fractions which consisted, respectively, chiefly of nuclei and coarse debris, mitochondria, microsomes, and final supernatant. Each fraction as well as an aliquot of the original homogenate was incubated at 37° C with progesterone. After 60, 30, or 5 minutes the incubation was ended and the residual progestin was extracted and bio-assayed. In general, most of the hormone had been inactivated, the liver nuclear fraction being the only one in which as much as 50–75% of the original hormonal activity consistently remained. Separate assay of the protein “bound” fraction revealed that “binding” was not a significant mechanism of inactivation. In vitro inactivation of progesterone by rat kidney as well as by rat liver was demonstrated.

The layers and cells of the early developing central nervous system lack direct counterparts in the adult and must be designated by a special terminology. The inconsistent and inaccurate language now in use leads to misunderstanding and a revision is proposed in which the four fundamental zones are termed the ventricular, subventricular, intermediate, and marginal zones. Each is defined according to the form, behavior, and fate of its constituent cells. All neurons and macroglia of the central nervous system can be derived from these developmental zones.

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.

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.

A description of chick duodenal morphogenesis is given. It is based on (a) quantitative measures of the change in size and shape of the duodenum, (b) a histogenetic study of the duodenal musculature, and (c) a quantitative description of changes in size, shape, and number of the duodenal villi throughout their development. Correlation of these data on a developmental time-scale has led to the following hypotheses: Mesenchymal cells are oriented by mechanical forces acting to distort a tissue. The orientation of smooth muscle is determined by the orientation of the premuscle mesenchyme in which it differentiates. In the wall of a hollow viscus such as the duodenum the contraction of the first layer of muscle to form will orient the remaining mesenchymal cells at right angles to itself, thus determining the orientation of the muscle-layers which subsequently differentiate. The duodenal villi are shaped by mechanical forces which act on the mucosa and which arise from epithelial expansion, on the one hand, and muscular contraction, on the other.

This investigation explored the relationships which exist between the growth of the lens and the growth of the outer eye coat (cornea plus sclera) during the development of the chick embryo eye. By introducing one end of a minute glass drainage tube through the eye wall at 4 days of incubation, vitreous substance was allowed to escape as it formed. Intubated eyes increased in overall size at rates which ranged from normal to very slow. Consequently, by 16 days of incubation the group of intubated eyes had achieved widely differing sizes, despite the fact that they were all of the same chronological age. In the first of two experiments, the total protein nitrogen per eye wall in the intubated eyes was proportional to the size which the eye had achieved by 16 days of incubation. This indicates that the smaller size of the outer eye coat reflected a depressed growth rate, and not simply a different spatial distribution of the same amount of substance, Furthermore, within the outer eye coat the growth rates of both the cellular portion, which was measured as total deoxyribonucleic acid per outer eye wall, and the extracellular portion, which was measured as hydroxyproline present in collagen per eye wall, were depressed. However, the amount of extracellular substance produced per cell (estimated as collagen/DNA) was constant regardless of the rate of growth of the outer eye wall. These results suggest that the enlarging vitreous body regulated the growth of the outer coat of the eye by controlling the number of cells in this layer rather than by controlling the amount of extracellular matrix produced by each cell. In a second experiment, similar to the first, the increase in size and the change in shape of lenses in intubated eyes were measured as a function of age. Such lenses grew normally and maintained normal shapes as development proceeded. Thus the growth of the lens was uncoupled from the growth of the outer eye coat. While the outer coat of the eye did not control the growth of the lens to harmonize with its own growth, the lens appeared to exert an indirect control over the growth of the outer coat of the eye in the following manner. Previous work had established that the lens must be present for vitreous substance to accumulate. The enlarging vitreous body, in its turn, helped control the growth of the outer coat of the eye.

The scleral ossicles, a ring of overlapping membrane bones, lie just outside the corneal margin in the eyes of domestic fowl. Eighty percent of the bony rings contain 14 bones; less than 1% have 13 bones; 19% have 15 bones; about 1% have 16 bones. Each bone is foreshadowed during development by a transient papilliform thickening in the overlying conjunctival epithelium. These conjunctival papillae appear on the eighth day of incubation and disappear on the twelfth day, when the corresponding preosseous membranes begin to ossify. Observations, and experiments involving the removal of specific papillae early or late in their maturation (in order to delete, or to reduce the size of, individual bones), demonstrated the following constraints on the morphogenesis of the scleral ossicular ring. (1) The number of ossicles is a function, not only of the number of papillae, but also of the distance between adjacent papillae; when two papillae lie close together, a single ossicle may arise beneath the pair. (2) There are three regions in the ring: nasal, dorsal (in both of which the bones overlap in one direction) and temporal (in which the bones overlap in the opposite direction). (3) The determinants of the direction of overlap between adjacent bones are extrinsic to the ossicles themselves and are distributed throughout each region, rather than confined to the discrete locations within each region where overlap normally occurs. (4) The three places in the ring where these regions meet are characterized by the loss (in 13-membered rings) or the addition (in 15- and 16-membered rings) of papillae and of their corresponding bones, and by transitions in the direction of overlap between the bones.

[3H]-retinol binding to cellular retinol-binding protein (CRBP) increased more than five-fold in developing chicken corneal epithelium at 14 days of incubation, the time when the initial increase in corneal transparency occurs in the developing embryo. In contrast, [3H]-retinoic acid binding to cellular retinoic acid-binding protein (CRABP) was highest at the earliest stages and decreased progressively to a very low level in the corneal epithelium near the time of hatching. Thus, there appear to be changing and differing requirements for retinol and retinoic acid during development of the chicken cornea.

The vertebrate eye focuses images of the environment in the plane of the retinal photoreceptors. Optimal visual function requires not only transparent dioptric media, but also that the relative sizes, shapes, and orientations of ocular tissues fall within the geometric tolerances imposed by the laws of optics. These functional requirements are met during embryonic development by the differentiation of highly specialized populations of cells from undifferentiated precursors and by the coordinated morphogenesis of the resulting tissues. Both processes are controlled to a remarkable extent by interactions which occur among the emerging tissues. Such interactions occur in an orderly, temporal sequence and help to mediate the developmental expression of one of the set of possible ocular phenotypic combinations defined by the individual genotype.

A piece of five-day embryonic lens epithelium was implanted in place of the right lens in each of 89 five-day-old chick embryos. The steps in the reconstitution of whole lenses from these grafts were identified by histologic analysis. Immediately following the implantation of the lens epithelium large numbers of cells came out of the anticapsular surface. Many of these died. Some of the survivors adhered in groups to the anticapsular surface of the implant. The cut edges of the implant became reflected so that the capsular surface was bent convexly. The reflected epithelium joined the groups of cells adhering to the anticapsular surface of the implant to form a flattened vesicle. The Golgi apparatuses of these cells lay at the anticapsular poles. The basal (capsular) poles of the cells always came to lie outermost regardless of the initial orientation of the implant. In time the vesicle filled the orifice of the eye cup and the reconstituted lens vesicle became invested externally by a continuous sheet of lens capsule. The cells in the wall of the vesicle that lay toward the vitreous body elongated to form lens fibers. A lens equatorial zone appeared facing the edge of the eye cup regardless of the orientation that had been given the implant initially. Thus, all of the reconstituted lenses were properly oriented in the eye.

When the lens of the 5-day chick embryo is surgically reversed so that its epithelium faces the neural retina the elongation of those lens cells which have already differentiated is arrested and the epithelial cells differentiate into a new set of lens fibers. This internal reorganization, together with a reversal in polarity at the lens equator, results in a complete reversal of the polarity of the-entire lens.

The embryonic chick corneal epithelium produces a highly structured acellular matrix beneath its basal surface during early development. This matrix, which contains collagen, serves as a morphogenetic template for subsequent stromal development in that the three-dimensional architecture of the adult corneal stroma is initially established, in miniature, in this epithelially derived connective tissue. Examination of the early corneal epithelium and matrix in both the light and electron microscope suggests that self assembly of the matrix may be one of several important factors in the morphogenesis of this early connective tissue.

Three hundred and forty whole mounts of retinas from domestic fowl aged two days of incubation to several months post-hatching were examined using a modified pyridine-silver technique. The first visible ganglion cell axons appeared near the posterior retinal pole between two and three days of incubation. These early axons grew immediately toward the optic stalk and became grouped in fascicles even before reaching it. The fascicles increased in number, diameter and length, became interconnected and became more parallel and orderly with age. The mean fascicle diameter and concentration increased with nearness to the optic fissure. Growth cones were seen only in the most vitread portion of the fiber layer. Two main groups of fascicles, one superficial and one deep, surrounded the head of the fissure by five days of incubation, each group entering the fissure at a different angle. At this time an arcuate pattern of fibers arose near the posterior pole of the retina. Fiber bundles were relatively thin and sparse in the center of this pattern. The distance between the fissure and the center of the pattern increased with age. The arcuate pattern virtually disappeared within several weeks, possibly indicating an abortive formation of a macula and fovea. The fibers at the periphery of the older retinas ran parallel to the iris, encircling but not entering it. These circumferential fibers, which first developed on the temporal side of the retina and later nasally, diverged from a point on the dorsal periphery of the retina. The relative position of this divergence point swung about 90° around the retinal periphery between five and one-half and eight days of incubation. Probable centrifugal fibers were first seen at 17 days of incubation.

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.

THE PURPOSE of this paper is to report the peculiar ocular histopathologic changes of an anomalous cleavage of the anterior chamber angle, choristomatous cornea-like tissue on the anterior iris surface, and a membrane in the chamber angle in an infant with multiple congenital anomalies. Report of a Case This 2-month-old white female infant died at St. Christopher's Hospital for Children on Jan 9, 1967 (autopsy: A-3-67; Wilmer Eye Pathology 29294). The infant's body was grossly malformed, exhibiting generalized asymmetry. The body weighed 2,124 gm (4 lb 10½ oz). The head was markedly flattened on the right side; the fontanel was located above the midportion of the left eyebrow rather than in the midline. The right eye bulged forward considerably. The pupil of the right eye was irregular, and the iris near the pupil had a rim of whitish tissue around it. The left eye was considered normal. The mandible

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.

A method is described for maintaining long term infusions of fluids into the chorioallantoic circulation of the embryo of the domestic fowl. Infusions can be continued up to 4 days in 6- to 17-day-old chicks, with subsequent hatching.

SummaryHomogenates of whole fetal mouse of the 12th, 13th, and 14th days of pregnancy and of fetal mouse liver of the 15th and 16th days inactivate progesterone in vitro.

Influence of Mouse Neural Retina on Regeneration of Chick Neural Retina from Chick Embryonic Pigmented Epithelium

Introduction The cornea of the adult vertebrate eye has a smaller radius of curvature than the posterior segment of the eye. In very early development, by contrast, the radii of curvature of the cornea and sclera tend to be equal. The manner in which the corneal curvature develops, and the forces responsible for it, are the concern of this paper. The interplay of a large number of factors is undoubtedly necessary for the normal development of the corneal bulge. The present effort, however, was confined to an investigation of the role of mechanical forces generated, on the one hand, by intraocular pressure and, on the other, by structural differentiation of the eye wall. Previous studies in this series 1,2 have demonstrated that these forces are responsible for the increase in size of the eye as a whole as well as for the normal expansion of the sclera, choroid coat, pigmented