AGE-DEPENDENT CHANGE IN THE TRANSDIFFERENTIATION ABILITY OF CHICK NEURAL RETINA IN CELL CULTURE*

We examined how the transdifferentiation ability of neural retinal cells into lens and/or pigment cells in call culture is changed with the development of the donor. Cells dissociated from neural retinas of chick embryos ranging from 3-day-old to the stage immediately before hatching and of 3-day-old chicks were cultured for about 60 days. The results clearly indicated that the transdifferentiation ability decreased with age. The latest developmental stage at which the differentiation of lens cells took place was in 18-day-old embryos. A gradual decrease in this ability was shown by the comparison of crystallin content in cultures prepared from embryos at different stages. The differentiation of pigment cells was recognized in cultures of neural retinas earlier than in 15-day-old embryos. Such loss of the ability of neural retinal cells to transdifferentiate into pigment cells earlier than into lens cells can be partially attributed to inhibitory factors accumulated in medium conditioned with many neuronal cells present in cultures.     

EFFECTS OF CULTURE MEDIA ON THE "FOREIGN" DIFFERENTIATION OF LENS AND PIGMENT CELLS FROM NEURAL RETINA IN VITRO

Neural retinal cells of 3.5-day-old quail embryos were cultured as a monolayer to examine their potentials for differentiation in vitro. The "foreign" differentiation into lentoid and pigment cells was much affected by the choice of medium (Eagle's MEM and Ham's F–12); in Eagle's MEM, neural retinal cells differentiated extensively into lentoid bodies and pigment cells, as previously reported in cultures of chick neural retinal cells, while in Ham's F–12, though the cells proliferated as well as in Eagle's MEM, the "foreign" differentiation is inhibited. When primary cultures were transferred to secondary cultures, the occurrence of "foreign" differentiation did not depend on the medium used for the primary culturing, but wholly on the medium used for secondary cultures. This difference in differentiation in two different media was quantitatively substantiated by measuring the amounts of α-, δ-crystallins and melanins of cultured cells.     

A decay of gap junctions in association with cell differentiation of neural retina in chick embryonic development.

Ultrastructural studies of thin-sectioned and freeze-cleaved materials were performed on developing retinal tissues of 3- to 9-day-old chick embryos to clarify the junctional structures between neural retinal cells and between neural retinal cells and cells of the pigmented epithelium. Frequency, size and position of gap junctions in developing neural retina are different at each stage of development. In 3-day-old embryos, some cells adhere to each other by gap junctions immediately below the outer limiting membrane of neural retinae. The size and number of gap junctions increase remarkably during 5-6 days of incubation. In this period of development, well developed gap junctions consisting of subcompartments of intramembrane particles are found between cell surfaces at both the outer limiting membrane region and the deeper portion of the neural retina. Gap junctions disappear thereafter, and at 7-5 days of incubation, small gap junctions are predominant between cell surfaces at the outer limiting membrane region, while the frequency of gap junctions in the deeper portion is very low. At 9 days of incubation, gap junctions are rarely found. Typical gap junctions are always found between neural retinal cells and those of the pigmented epithelium in embryos up to 7-5 days of incubation. Tight junctions are not found in the neural retina or between neural retina and pigmented epithelium throughout the stages examined.     

Ultracytochemical study on in vitro differentiation of neural retinal cells of chick embryos

In order to study cell differentiation and morphogenesis of neural retina, ultracytochemical examination for acetylcholine esterase (AChE) was carried out on neural retinal cells from 6-day-old chick embryos cultured in monolayer for 20 days. AChE is a suitable marker for identifying cell specificity and structure of cultured neural retinal cells, because its specific localization in the intact chick neural retina has been established. After about 2 weeks of culturing a number of cell aggregates formed on the monolayer sheet of glial cells, in which cell bodies were generally located on the periphery regions while their cellular processes were in the center, forming neuropil structures. Among such peripherally located cells presumptive ganglion, amacrine, bipolar, and photoreceptor cells could be distinguished. In the neuropil structures, some cellular processes had typical ribbon synapses indicating that these structures correspond to the plexiform layers of the retina. We could also classify the neuropils into two types of both from the AChE activity and from the structure of the nerve terminals. These findings indicate that our cell culture system can be used for the study of cell differentiation and histogenesis of retinal cells.     

ULTRASTRUCTURAL STUDY OF THE PROCESS OF ROSETTE FORMATION BY DISSOCIATED NEURAL RETINAL CELLS IN STATIONARY CULTURE

Well orientated two-dimensional rosettes were produced in stationary cultures of dissociated neural retinal cells of 5 1/2-day-old chick embryos. Observations by electron microscopy were made on aggregates at the different steps in the process leading to rosette formation. Though all of the dissociated neural retinal cells showed a clear morphological polarity, primary cell-to-cell adhesion occurred at random with respect to the cellular polarity. A special junction of the zonula adhaerens type was found at the contact area between the "mitochondria-containing portions" or their neighboring regions of both adjoining cells. During stationary culturing, the contact area between the cells gradually increased. Where the "mitochondria-containing portions" of adjoining cells were brought into contact with each other, junctions of zonula adhaerens type were formed between them. In this way, a radial arrangement of all the cells within an aggregate was established, leading to the formation of the fundamental structures of rosettes.     

Astrocytes induce neural microvascular endothelial cells to form capillary-like structures in vitro

Astrocytes maintain a unique association with the central nervous system microvasculature and are thought to play a role in neural microvessel formation and differentiation. We investigated the influence of astroglial cells on neural microvascular endothelial differentiation in vitro. Using an astroglial-endothelial coculture system, rat brain astrocytes and C6 cells of astroglial lineage are shown to induce bovine retinal microvascular endothelial (BRE) cells to form capillary-like structures. Light microscopic evidence for endothelial reorganization began within 48 hours and was complete 72-96 hours following the addition of BRE cells to 1-day-old astroglial cultures. The extent of BRE reorganization was quantitated by computer-assisted analysis and shown to be dependent upon the density of both the BRE and C6 cells within the cocultures. Coculture conditions in which BRE cells were separated from C6 cells by porous membranes failed to generate this endothelial cell change. Likewise, C6-conditioned media and C6-endothelial coculture conditioned media did not induce BRE cell reorganization. Extracellular laminin within the C6-endothelial cocultures, identified by indirect immunofluorescence, was concentrated at the endothelial-astroglial interface of capillary-like structures consistent with incipient basement membrane formation. Astroglial cells accumulated adjacent to capillary-like structures suggesting the presence of bidirectional influences between the reorganized endothelial cells and astroglia. This is the first demonstration of astroglial induction of angiogenesis in vitro and these findings support a functional role for perivascular astrocytes in the vascularization of neural tissue such as retina and brain.     

THE DIFFERENTIATION OF PIGMENT CELLS IN CULTURES OF CHICK EMBRYONIC NEURAL RETINAE

Neural retinal cells of 8–9 day-old chick embryos were differentiated into pigment cells in the conditions of cell culture for about 25 days. The increase of pigment cells in vitro was semi-quantitatively shown, by counting the number of black foci of pigmented cells per plate throughout the culture period. The increase paralleled the increase in the activity of tyrosinase. The addition of a small number of pigment cells freshly dissociated from tapeta to the cultures of neural retinae did not increase the number of black foci in vitro . Electron microscopic observations revealed the morphological differences of melanin granules between those in pigment cells of the neural retinal cultures and those in cultured tapetum cells. It was discussed that pigment cells appearing in the neural retinal cultures were derived from neural retinal cells, but not from contaminated cells of the tapetum.     

The differentiation of the nerve and glial cells of neocortical transplants placed into the brain of adult rats

Embryonal neural tissue of 17-day-old rat embryos was transplanted into the brain of adult Wistar rats to test the differentiation of transplants with reference to the normal cerebral cortex development. The control and the experimental rats were decapitated 2, 5, 7, 10, 15, 20, 25, and 35 days after the transplantation. Differentiation of neural tissue was studied using monoclonal antibodies against neurofilaments as well as by counting the proportion of differentiated neurons. The glial differentiation was studied by immunohistochemical method using monoclonal antibodies against acid glial fibrillar protein and vimentin. The differentiation of neural cells of transplants proved to be synchronous with the normal ones while the differentiation of glial cells accelerates.     

DIFFERENTIATION AND TRANSDIFFERENTIATION IN VITRO OF NEURAL RETINA FROM MUTANT CHICKENS WITH HYPERPLASIC LENS EPITHELIUM1)

Mutant chickens, Hy-1 and Hy-2, show abnormalities in growth and differentiation of the lens epithelium. In this study, neural retinal cells (NR cells) from 3.5-day-old embryos of these mutants were cultured, and the differentiation in vitro was compared with the cells of the normal strain. Hy-1 cells in vitro were characterized by a delay in the first appearance of neuronal cells (N-cells) and by excessive production of this cell type at later stages. By contrast, the Hy-2 cells were indistinguishable from the normal cells in the early phase of culturing. In spite of the marked difference of Hy-1 NR cells in neuronal differentiation up to about 7 days in culture, the transdifferentiation of lens and pigmented cells occurred to a similar extent and with the same time schedule as cultures of normal cells. A number of lentoid bodies were formed by about 10 days. The relative composition of the three major classes of crystallins in transdifferentiated lens cells was almost identical between normal and Hy-1 strains. The results were discussed in comparison with the previous results of cell culture of NR of 8-day embryonic mutant chickens, and it was concluded that the process of transdifferentiation in cell culture is different between NR from 3.5-day-old and 8-day-old embryos.     

Function of Rx, but not Pax6, is essential for the formation of retinal progenitor cells in mice

Rx plays a critical role in eye formation. Targeted elimination of Rx results in embryos that do not develop eyes. In this study, we have investigated the expression of Otx2, Six3, and Pax6 in Rx deficient embryos. We find that these genes show normal activation in the anterior neural plate in Rx-/- embryos, but they are not upregulated in the area of the neural plate that would form the primordium of the optic vesicle. In contrast, in homozygous Small eye embryos that lack Pax6 function, Rx shows normal activation in the anterior neural plate and normal upregulation in the optic vesicle/retinal progenitor cells. This suggests that neither Rx expression nor the formation of retinal progenitor cells is dependent on a functional copy of the Pax6 gene, but that Pax6 expression and the formation of the progenitor cells of the optic cup is dependent on a functional copy of the Rx gene.     

Enhancement of differentiation of lens and pigment cells by ascorbic acid in cultures of neural retinal cells of chick embryos.

When dissociated cells of neural retinae of 9-day-old chick embryos were cultured in Eagle's minimum essential medium supplemented with dialyzed fetal calf serum, both the proliferation and differentiation of the neural retinal cells were inhibited. These cells remained quiescent and flattened. When ascorbic acid was added to such a medium, the cells started to grow and differentiated into lentoid bodies and pigmented cells after about 10 days.     

An antibody to a receptor for fibronectin and laminin perturbs cranial neural crest development in vivo

Previous studies from this laboratory (M. Bronner-Fraser (1985). J. Cell Biol. 101, 610) have demonstrated that an antibody to a cell surface receptor complex caused alterations in avian neural crest cell migration. Here, these observations are extended to examine the distribution and persistency of injected antibody, the dose dependency of the effect, and the long-term influences of antibody injection. The CSAT antibody, which recognizes a cell surface receptor for fibronectin and laminin, was injected lateral to the mesencephalic neural tube at the onset of cranial neural crest migration. Injected antibody molecules did not cross the midline, but appeared to diffuse throughout the injected half of the mesencephalon, where they remained detectable by immunocytochemistry for about 22 hr. Embryos were examined either during neural crest migration (up to 24 hr after injection) or after formation of neural crest-derived structures (36-48 hr after injection). In those embryo fixed within the first 24 hr, the major defects were a reduction in the neural crest cell number on the injected side, a buildup of neural crest cells within the lumen of the neural tube, and ectopically localized neural crest cells. In embryos allowed to survive for 36 to 48 hr after injection, the neural crest derivatives appeared normal on both the injected and control side, suggesting that the embryos compensated for the reduction in neural crest cell number on the injected side. However, the embryos often had severely deformed neural tubes and ectopic aggregates of neural crest cells. In contrast, several control antibodies had no effect. These findings suggest that the CSAT receptor complex is important in the normal development of the neural crest and neural tube.     

Embryonic chicken retinal cells can regenerate all cell layers in vitro,but ciliary pigmented cells induce their correct polarity

Summary The capacities of retinal and pigmented cells to regenerate histotypic in-vitro-retinae (IVR) in rotary culture were investigated by dividing the eye cups of 6-day-old chicken embryos into a central and a peripheral part; they were cut along the ora serrata, and the retinal and the pigmented constituents of both parts were isolated. The 4 dissociated cell populations were cultured separately and in all double combinations. Two different types of IVR's were generated; one developed from central or peripheral retinal cells, the other required the addition of pigmented cells from the ciliary margin of the eye. The shape of these IVR's was examined using scanning electron microscopy, and they were also characterized histologically. The acetylcholinesterase pattern marked the inner half of the retina; F11-antibody and a peanut agglutinin marker revealed both plexiform layers and a radial fiber system. In both types, organized histotypical areas consisted of complete sets of retinal layers. In the type containing pigmented cells from the eye periphery, the sequence of layers was identical with that of an in-situ-retina (laminar IVR). In IVR's derived from retinal cells only, the sequence of layers was reversed (rosetted IVR).     

The plakin Short Stop and the RhoA GTPase are required for E-cadherin-dependent apical surface remodeling during tracheal tube fusion

Cells in vascular and other tubular networks require apical polarity in order to contact each other properly and to form lumen. As tracheal branches join together in Drosophila melanogaster embryos, specialized cells at the junction form a new E-cadherin-based contact and assemble an associated track of F-actin and the plakin Short Stop (shot). In these fusion cells, the apical surface determinant Discs Lost (Dlt) is subsequently deposited and new lumen forms along the track. In shot mutant embryos, the fusion cells fail to remodel the initial E-cadherin contact, to make an associated F-actin structure and to form lumenal connections between tracheal branches. Shot binding to F-actin and microtubules is required to rescue these defects. This finding has led us to investigate whether other regulators of the F-actin cytoskeleton similarly affect apical cell surface remodeling and lumen formation. Expression of constitutively active RhoA in all tracheal cells mimics the shot phenotype and affects Shot localization in fusion cells. The dominant negative RhoA phenotype suggests that RhoA controls apical surface formation throughout the trachea. We therefore propose that in fusion cells, Shot may function downstream of RhoA to form E-cadherin-associated cytoskeletal structures that are necessary for apical determinant localization.     

Demonstration of Transdifferentiation of Neural Retina from Pigmented Retina in Culture1

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.     

Suppression of lens growth by alphaA-crystallin promoter-driven expression of diphtheria toxin results in disruption of retinal cell organization in zebrafish

In order to study lens-retina relationships during development, we cloned the zebrafish alphaA-crystallin cDNA and its promoter region. Using a 2.8-kb fragment of the zebrafish alphaA-crystallin promoter (z(alpha)Acry), we expressed the diphtheria toxin A fragment (DTA) in zebrafish embryos in a lens-specific manner. Injection of the z(alpha)Acry-DTA plasmid into eggs at the one-or two-cell stage resulted in the formation of small eyes, in which both lens and retina were reduced in size. In the DTA-expressing lenses, their fiber structure was disorganized, indicating that normal lens development had been abrogated. The neural retina also showed abnormal development, although this tissue did not express DTA. Lamination in the retina did not develop well, and molecular markers for the outer and inner plexiform layers were either abnormally expressed or absent. However, cell type-specific markers of ganglion and bipolar cells, as well as photoreceptors, were expressed in appropriate positions, indicating that initial differentiation of these retinal subpopulations occurred in the DTA-expressing embryos. Cell proliferation also proceeded normally in these embryos, although apoptosis was enhanced. These results suggest that the differentiated lens plays a critical role in the morphogenetic organization of retinal cells during eye development in zebrafish embryos.     

Differentiation of Lens and Pigment Cells in Cultures of Brain Cells of Chick Embryos

Dissociated cells of brains (tel- and diencephalons) of 3.5-day-old chick embryos were cultivated in vitro under the cell culture conditions which are known to be permissive for neural retinal cells (NR cells) to transdifferentiate into lens and/or pigmented epithelial cells (PE cells). The differentiation of lentoid bodies (LBs) with lens-specific (δ-crystallin and PE cells with melanin granules was observed in such brain cultures.
LBs appeared in two different phases, i. e., 2–3 days and 16–30 days of cultivation, and after 40 days of culture these structures were formed in all 60 culture dishes. Sometimes, LBs were observed in foci of PE cells formed during earlier stages of brain cultures. When similar brain cultures were prepared with older embryos of 5-, 8.5-, 14-, and 16-days of incubation, no differentiation of lens and PE cells was observed.     

Light-induced acceleration of retina development in a mouth-brooding teleost.

Rearing animals in the dark had been shown to be either without effect on the development of the retina or to result in a reduction or a delay of retinal maturation. In the present study, the influence of light on a retina which normally develops under conditions of very dim light has been investigated. When 3-day-old embryos of the mouth-brooding teleost Tilapia leucosticta are placed into a continuously lighted environment, from day 6 on, optic tract diameter and inner plexiform layer thickness increase up to day 10 or 12. In the dark-reared retina, this increase occurs only after day 10. Similarly, synaptic junctions of the inner plexiform layer appear at about day 6 in the light-reared retina and increase continuously on following days, whereas in the retina of embryos reared in darkness, they appear at day 7 and do not increase essentially before day 11. These and other data suggest that continuous light induces a precocious growth of retinal structures. The first differences between light- and dark-reared retinae appear synchronously with the beginning of receptor cell development and prior to the first synaptic junctions. A non-neurally mediated effect of light on the retinal ganglion cells is consequently assumed.     

An ethanolic phosphotungstic acid (EPTA) analysis of photoreceptor and synaptic ultrastructure in the guinea pig retina

Ethanolic phosphotungstic acid (EPTA) has been used to elucidate the structure of certain organelles contained within retinal cells not clearly discernible using conventional preparations. Both synaptic and nonsynaptic components of the guinea pig neural retina have been analyzed. Within the photoreceptor (PR) cell EPTA-stained components include the connecting cilia, their basal bodies, and the root filament system. Cross-striated fibrillar organelles, similar in appearance to the root filaments, are also observed in the nuclear region, the synaptic terminal and other parts of the PR cell. The possible structural continuity and significance of these structures are discussed. Within retinal synapses of both the inner and outer plexiform layers, ribbons and associated paramembranous specializations are stained. The photoreceptor ribbons have a trialaminar structure with filamentous, tufted borders. Synaptic cleft material and postsynaptic densities are also stained. Bipolar cell synapses in the inner plexiform layer contain stained short ribbons as well as closely associated peg-like densities extending towards the presynaptic membrane.     

The possibility of lens formation in explants of the retina and pigment epithelium of the eye in chick embryos

Undissociated tissue explants of the retina and retinal pigment epithelium (RPE) of 3,5-, 4-, 5- and 8-day-old chick embryos were cultured in vitro. After 7 days in culture, lentoids were observed in explants of either retina or RPE from 3,5-, 4- and 5-day-old embryos. As demonstrated by immunohistochemistry, these lentoids contained specific chick lens proteins (alpha-, beta- and delta-crystallins). No crystallin-containing cells were found in eye tissue explants from 8-day-old embryos. However, when 5-bromo-deoxyuridine (25 microM) was introduced into the medium at the beginning of culturing (for 12 h), large eosinophilic cells containing alpha-, beta- and delta-crystallins were detected in retinal explants of the 8-day old embryos. Thus, retina and RPE of 3,5-5-day-old chick embryos are capable of lens differentiation after explantation in vitro without dissociation into individual cells. This capacity is lost during development.