Chick Embryo Eyes Research Articles

Preliminary characterization of cell surface-extracellular matrix linkage complexes in cultured retinal pigmented epithelial cells

In this report, we describe the relative distribution of vinculin, talin, and fibronectin in cultured retinal pigmented epithelial cells from chick embryo eyes. We show that in these cells vinculin is present in both focal cell-substratum and cell-cell contacts, whereas talin is present only in the cell-substratum contacts. When cells are double-labeled for talin and fibronectin and viewed at the substratum level, fibronectin is not detectable and talin is concentrated in plaques corresponding to focal contacts. However, when the same cells are viewed at the apical level, both talin and fibronectin are present in a fibrillar pattern. In addition to fibrils which are both talin- and fibronectin-positive, there are areas which are either talin-positive and fibronectin-negative or, vice versa, talin-negative and fibronectin-positive. These observations indicate an interesting variability in the composition of transmembrane linkages in retinal pigmented epithelial cells in vitro.

Anterograde tracing of retinal axons in the avian embryo with low molecular weight derivatives of biotin

Several reactive biotin esters were injected into the eyes of chick and quail embryos at various stages of development. Four of the biotin esters reacted with molecules of the eye tissue and were detected with light and electron microscopy in fluorescein isothiocyanate and peroxidase-avidin incubated sections and whole mounts. Intra and extracellular components of the lens, the vitreous body, and the retina were labeled to different degrees. Three of the biotin esters (biotin- N-hydroxysuccinimidester, biotin-ϵ-aminocaproic acid- N-hydroxysuccinimidester, and desthiobiotin- N-hydroxysuccinimidester) prominently marked the optic fiber layer in the retina and the biotin labels were transported along the optic pathway. The tracers were detected up to the growth cone of axons 24 to 36 hr after injection. Explants from biotin marked retinas were cultured on collagen or basal laminae. During culturing axons grew out from these explants into the substratum showing that labeled tissue and nerve fibers were viable. The development of the optic pathway at the chiasma of quail embryos was studied using the biotin/avidin tracing. The bulk of fibers emerging from the retina crossed as shown by double labeling of both optic nerves in a complex pattern of segregated and interdigitizing axon bundles at the chiasma toward the contralateral side of the brain. From stage 25 onward a minor ipsilateral projection was found. At the same developmental stage a few fibers traveled into the contralateral optic nerve and grew retrogradely toward the contralateral eye. The percentage of specimens having this retino-retinal projection increased during development from 53% (stage 24 to 27; E3.5–E5.5) to 89% (stage 29 to 35; E6–E8) and declined to 40% at late embryogenesis (stage 37 to 41; E9–E12). The fact that all retinal axons were found within predictable pathways with some of them running in the wrong direction suggests that nerve fiber pathways provide accurate positional information, but at best weak directional information for growing nerve fibers.

Insulin and IGF receptors are developmentally regulated in the chick embryo eye lens

We have previously reported that insulin-like growth factor (IGF) receptors appear to predominate over insulin receptors in early stages of embryogenesis in the chick (days 2–3 whole embryo membranes). Overall, [ 125I]IGF I and II binding to specific receptors was maximal when the rate of brain growth is highest. In the present study we used the embryonic chick lens, a well-defined tissue composed of a single type of cell, to analyse whether changes of insulin and IGF I binding are correlated with changes in growth rate and differentiation state of the cells. We show that both insulin receptors and IGF receptors are present in the lens epithelial cells, and that each type is distinctly regulated throughout development. While there is a direct correlation between IGF-binding capability and growth rate of the cells, there is less relation to differentiation status and embryo age. Insulin receptors, by contrast, appear to be mostly related to the differentiated state of cells, decreasing sharply in fibers, irrespective of their developmental age.

Localization of laminin, type IV collagen, fibronectin, and heparan sulfate proteoglycan in chick retinal pigment epithelium basement membrane during embryonic development.

Type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin were localized in the basement membrane (BM) of chick retinal pigment epithelium (RPE) during various stages of eye development. At different times over a 4-17 day period after fertilization, chick embryo eyes were dissected, fixed in periodate-lysine-paraformaldehyde, and 6 micron frozen sections through the central regions of the eye were prepared. Sections were postfixed in -20 degrees C methanol and stained immediately by indirect immunofluorescence using sheep anti-mouse laminin, sheep antimouse type IV collagen, rabbit anti-mouse heparan sulfate proteoglycan, and mouse monoclonal anti-porcine plasma fibronectin. Fluorescein-labeled F(ab')2 fragments of the appropriate immunoglobulins (IgGs) were used as secondary antibodies. Laminin could be readily demonstrated in the BM of the RPE during all stages of development. The staining for type IV collagen, fibronectin, and heparan sulfate proteoglycan HSPG) was less intense than that for laminin, but was also localized in the BM along the basal side of the RPE. In addition to staining the BM, antiserum to HSPG, gave a diffuse labeling from day 9 onward, above the RPE extending into the region of the photoreceptors. Whereas the intensity of staining generally increased between day 4 and day 17 of development, the distribution of the different BM components did not change. Hence the presence of type IV collagen, laminin, fibronectin, and HSPG in the BM of RPE in vivo during all the stages of development investigated supports the concept that these macromolecules are important basic components of this, and other, BMs. Furthermore, these results indicate that the composition of the BM of RPE cells in vivo is similar to the BM material deposited by RPE cells in vitro (Turksen K, Aubin JE, Sodek JE, Kalnins VI: Collagen Rel Res, 4:413-426, 1984) and that the in vitro cultures can therefore serve as a useful model for studying BM formation.

The focal adhesions of chick retinal pigmented epithelial cells.

Retinal pigmented epithelial (RPE) cells obtained from the eyes of chick embryos from colonies in vitro in which cells at the periphery of the colony express an undifferentiated, well-spread morphology and develop extremely large areas of cell-substratum adhesion. These adhesions can be classified as focal on the basis of the following: (a) their black surface reflection interference image, the contrast of which is not affected by changes in either the wavelength of the incident light or the refractive index of the immersion medium; (b) their association with the termini of actin-containing microfilament bundles; and (c) their ability to be labelled with antiserum against vinculin, a protein specific for adhesions of the focal type. The focal adhesions of RPE cells comprise laterally associated individual focal contacts, the mechanism by which this association is achieved and maintained is yet unknown. Because of the unusually large size and excellent microscopical definition of their focal adhesions, I used RPE cells to investigate the role of other actin-associated proteins in adhesion complexes. One of these, nonerythroid spectrin (fodrin), a protein suggested to play a role in anchoring actin filaments to the plasma membrane, was neither concentrated in nor excluded from the focal adhesions of RPE cells. Thus, at least in this cell type, spectrin seems unlikely to serve as a link between the major actin-containing microfilament bundles and the plasma membrane in the regions of cell-to-substratum contacts.

Asymmetrical developmental pattern of uptake of lucifer yellow into amacrine cells in the embryonic chick retina

Whole chick embryo eyes between embryonic day 10.5 and 12 (E10.5–E12) were incubated in a solution of Lucifer Yellow and examined in frozen sections. Starting around day 10.5, brightly stained cells were observed in the innermost part of the inner nuclear layer. Their size, shape and location suggest that most of them represent a subclass of amacrine cells. A distinct spatio-temporal development of Lucifer Yellow-staining of this subpopulation of cells within the inner nuclear layer between E10.5 and E12 is revealed in detail using 3-dimensional models of Lucifer Yellow-stained eyes. The staining can be first observed at a specific location at the ventro-posterior side not far from the fundus. It then spreads radially in a complex pattern reaching the ora serrata first on the ventro-posterior side, then sequentially reaching into the dorso-posterior, the ventro-anterior and finally the dorso-anterior quadrants of the retina. Our results suggest that a horizontal (posterior-anterior) axis and a vertical (ventro-dorsal) axis function as a coordinate system during differentiation of the tissue. We conclude that there are precise spatio-temporal patterns of Lucifer Yellow-staining which most probably reflect spatio-temporal patterns of cell differentiation within the chicken retina. The correlation of these findings with other data on spatio-temporal patterns during differentiation of the chicken retina is discussed.

Selective uptake of Lucifer Yellow into different cell populations of the developing chicken retina

Whole chick embryo eyes at different stages of development were incubated in a solution of Lucifer Yellow (LY) and examined in frozen sections. Different cell populations selectively take up the dye during the course of development: from embryonic day 4 – day 8 of incubation (E4-E8) only a subpopulation of neuroepithelial cells are stained, which at E7-E8 are located mainly near the periphery between the prospective ora serrata and the outer edge of the retina. This staining disappears after E8. A second phase starts at E10, with a weak but specific staining of a small population of cells in the outer nuclear layer which becomes less pronounced by E12. Starting around E10.5 a bright staining of cells in the innermost part of the inner nuclear layer can be observed. Their size, shape and location suggest that most of them represent a subclass of amacrine cells (including all ‘displaced amacrine cells’). Beginning at E15, the staining pattern becomes more complex. A single cell analysis of the percentage of LY-stained cells which are stained either before or after papain-treatment and dissociation of the intact tissue confirms and extends the histological findings.

The origin of the ectomesenchymal condensations which precede the development of the bony scleral ossicles in the eyes of embryonic chicks

The origin of neural crest-derived ectomesenchymal condensations located subjacent to scleral papillae and the development of the more deeply situated scleral ossicles were investigated in scleral tissues explanted from the eyes of chick embryos at Hamburger & Hamilton (1952) stages 30-38 (6 1/2-12 days of incubation). Explants were pulse labelled with [3H]thymidine for 4h in vitro. Nuclear counts and % labelling indices were calculated for standardized areas within and between the condensations. At all stages exhibiting condensations, the % labelling indices were higher within the condensations than in tissues between condensations. % labelling declined with maturity, but the decline was greater between than within condensations. Regional differences in intensity of proliferation in the ectomesenchyme centred about the scleral papillae seemed to be the best explanation for the development of these condensations. The condensations disappeared concomitant with the complete degeneration of the adjacent papillae. A new distribution of labelled nuclei 70-100 micrometer deep in the tissue and beneath the original sites of the condensations preceded the appearance of ossicle primordia. The roles of the scleral papillae, the ectomesenchymal condensations and deeper primordia in the development of scleral ossicles are discussed.

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.

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.

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

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.

Lens development: III. Relationship between the growth of the lens and the growth of the outer eye coat

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.

Development of the retinotectal topographic projection in the chick embryo

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.

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

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.

Growth of lens epithelium in culture. I. Characteristics of growth.

The study of lens epithelium in tissue culture was started by Daniel B. Kirby in 1926. He observed the differentiation of the epithelium into lens fibers when the anterior one-third of 52-hour-old chick embryo eyes were cultured in vitro. In 1929 Kirby, Estey, and Tabor found that cultures of chick embryo lens epithelium were more successful in the regular culture media than when the various constituent inorganic salts were increased more than 25% over the normal. They also found an irregular effect of hypertonic media on these cultures, while a 10% to 15% dilution had a marked depressant action on the growth. When they used a slightly more alkaline medium (pH 8.2), they observed a fairly marked depression of the rate of growth in the cultures. In 1932 Kirby, Estey, and Weiner observed that an increase in the glucose content of the medium produced no real effect on the growth

A PURE STRAIN OF CARTILAGE CELLS IN VITRO

1. A strain of cartilage cells, obtained from the pars cartilago sclerae of the eye of chick embryos, has been cultivated for more than 3 months in vitro. 2. The initial growth of the cartilage was possible only on the free surface of the coagulum. 3. The hyaline substance disappeared during cultivation in vitro. The succeeding stages of a transformation from small, lymphocyte-like cells into large, spindle-shaped cells were observed. The cartilage cells were spindle-shaped and grew in close contact, forming thin membranes. In surface-grown cartilage cells, the nucleus, usually containing one large nucleolus, stained less deeply than the cytoplasm. 4. The rate of growth of cartilage was slower than that of fibroblasts and epithelium. After cultivation on the surface of the coagulum, the cartilage cells could multiply even when embedded in the coagulum. But their growth was less extensive and uniform.