Cell division and cellular morphology of the chick retinal pigmented epithelial cells in culture: a time-lapse analysis (original) (raw)
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Neuroscience Letters, 1984
We report here that, in comparison to aggregates from retinal cells alone, addition of pigmented epithelial cells to retinal cells in rotary culture results in a pronounced increase of spatial order. A particularly high level of organization is found in about 15-20°70 of the aggregates. In these 'retinoids' the main layers characteristic of developing in vivo retinae can be distinguished in correct sequential arrangement on the basis of morphological criteria and by using acetylcholinesterase histochemistry [5, 6, 15], peanut agglutinin-lectin binding [11] and Lucifer Yellow staining [7-9]. Dissociated single cells from embryonic chick retinae reaggregate quickly and form relatively organized histotypic structures in a rotary culture system. Reaggregation of dissociated single cells starts with a random alignment of retinal cells, followed by the formation of primary rosettes and by the incorporation of additional cells into the aggregate [1, 4, 10-14]. We studied the internal order of these aggregates upon prolonged incubation of 2-3 weeks. Fujisawa [3] described specific layers which are formed upon aggregation of retinal cells on the chorioallantoic membrane. We were able to demonstrate a similar high degree of laminar organization in a pure in vitro system of aggregates derived from mixed cultures of retinal and pigmentepithelial (PE) cells. Methodologically, the neuroretinae and pigmented epithelia from E6 chicken embryos (White Leghorn) were dissected and washed with Hank's solution. Pigmented epithelia were first incubated in 1 mg/ml collagenase and 300 U/ml hyaluronidase (both from Boehringer) in Eagle's Minimal Essential Medium (Ea-MEM) for 10 min at 37°C, washed once in Hank's solution and treated with 1 mg/ml trypsin (Difco) for 20 min at 37°C. Isolated retinae were digested only by trypsin (10 min, 20°C and 10 min, 37°C). Then the tissues were rinsed in Ea-MEM and mildly dissociated into single cells in the presence of 0.05 mg/ml DNAase. After a 3-fold wash in Ea-MEM the cells were resuspended in aggregation medium (10070 fetal calf serum, 2°70 chicken serum, 1 070 glutamine, 0.1 070 penicillin/streptomycin and 0.02 mg/ml gentamycin in Ea-MEM). A 2 ml suspension of cells consisting either of 2-5 x 106 retina
Experimental Eye Research, 1988
Embryonic retinae from 541-day-old chicks (E5-E6) were cut into stripes either in close contact with (RPE stripes) or in absence of the neighboring retinal pigmented epithelium (R stripes). The stripes were explanted and cultivated in vitro for up to 6 days, during which time they show the following differences in their characteristics of growth and differentiation. Compared with R stripes, RPE stripes morphologically showed a significant increase in size during the first 2 days in culture. Using E5 tissue, this is also demonstrated by a higher rate of cell proliferation (as measured by uptake of radioactive thymidine as well as by DNA contents). In contrast, R stripes after two days in culture show a much stronger neurite growth. After longer periods of culturing (5-6 days) we can show by cholinesterase histochemistry (ACHE and BChE) and by PNA-lectin binding that the RPE stripes have started to form all major layers of the in vivo retina, whereas R stripes remain unstratified and start to degenerate earlier. We conclude that the pigment epithelium might exert a specific stimulus on growth and tissue differentiation of the neural retina not only during in vitro, but possibly also during in vivo development. The in vitro methods introduced here could become useful model systems to further investigate the significance of the RPE for developmental, regenerative and even adult processes of the neural retina. Their future applicability in ophthalmologic research is briefly discussed.
Neuroscience Letters, 1986
Key words." chick retina aggregate-lamination pigment epithelium Wc report here a striking age dependency for tissue reconstruction capacity in rotatmg cultures of chick retinal cells, in particular in mixtures of retinal and pigment epithelial cells. The comparison of sections after acetylcholinesterase histochemistry delivered the following results. (1) In aggregates from retinal cells, sorting out is clearly observable, but only a minor age-dependent effect is detectable, e.g. the laminar order of aggregates derived from 6-day-old cells is increased compared to aggregates from other stages. (2) In aggregates from mixtures of retinal and pigment epithelial cells, the degree of laminar order is highest with cells from 5-day-old embryos and decreases significantly at older ages. The highest number of aggregates with the reconstruction of all main retinal layers was obtained by the reaggregation of retinal cells of embryonic day 5 (25%). (3) To achieve laminar order in aggregates, at all ages, the addition of pigment epithelial cells seems to be a prerequisite.
Differentiation of Lens and Pigment Cells in Cultures of Brain Cells of Chick Embryos
Differentiation, 1982
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 andor pigmented epithelial cells (PE cells). The differentiation of lentoid bodies (LBs) with lens-specific d-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. K. Nomura: Lens Differentiation in Brain Cell Cultures 33. Yamada T (1977) Control mechanisms in cell-type conversion in newt lens regeneration. In: Wolsky A (ed) Monographs Develop Biol, Vol. 13. Karger. Basel, pp 1-126 34. Yasuda K (1979) Transdifferentiation of 'lentoid' structures in cultures derived from pigmented epithelium was inhibited by collagen. Develop Biol 68: 618-623 35. Yasuda K, Eguchi G, Okada TS (1981) Age-dependent changes in the capacity of transdifferentiation of retinal pigment cells as revealed in clonal cell culture. Cell Differentiation 10: 3-11
Proceedings of the Royal Society B: Biological Sciences, 1997
Reaggregation of dispersed retinal cells of the chick embryo leads to histotypic retinospheroids in which the laminar organization remains incomplete: photoreceptors form rosettes which are surrounded by constituents of the other retinal layers. Here, for the ¢rst time, a complete arrangement of layers is achieved in cellular spheres (stratoids), provided that fully dispersed retinal cells are younger than embryonic day E6, and are reaggregated in the presence of a monolayer of retinal pigmented epithelium (RPE). A remarkable mechanism of stratoid formation from 1 to 15 days in vitro is revealed by the establishment of a radial MÏller glia sca¡old and of photoreceptors. During the ¢rst two days of reaggregation on RPE, rosettes are still observed. At this stage immunostaining with vimentin and F11 antibodies for radial MÏller glia reveal a disorganized pattern. Subsequently, radial glia processes organize into long parallel ¢bre bundles which are arranged like spokes to stabilize the surface and centre of the stratoid. The opsin-speci¢c antibody CERN 901 detects photoreceptors as they gradually build up an outer nuclear layer at the surface. These ¢ndings assign to the RPE a decisive role for the genesis and regeneration of a vertebrate retina.
Seminars in Cell & Developmental Biology, 1998
Inductive effects of the retinal pigmented epithelium (RPE) on histogenesis of the avian retina as revealed by retinospheroid technology @ Paul G. Layer, Andrie Rothermel and Elmar Willbold During eye formation, inductive phenomena occurring between retinal pigmented epithelium @P@ and retina are not well understood. After briefly summarizing the normal development of retina and RPE, we present three-dimensional in vitro models of the chick embryonic retina which allows elucidation of RPE-retina interactions. In such retinospheroids, a complete arrangement of layers is achieved, provided that dispersed retinal cells are: (I) young enough; and (2) reaggregated on a monolayer of RPE. Thereby, the RPE extends cell prolifration, while differentiation is much delayed. These findings assign to the RPE a decisive role for the genesis and regeneration of a vertebrate retina.
Scanning electron microscopic studies on the development of the chick retina
Cell and Tissue Research, 1976
Fixed retinae of chick embryos and chicks of the first week after hatching were fractured and examined with the scanning electron microscope. The matrix cells of the retina proliferate up to the beginning of the second week. The migrating cells are oriented in cell cords. This columnar organization prevails up to the development of the plexiform layers formed as a consequence of the outgrowth of the dendritic and axonal cell processes. Special attention was paid to the differentiation of the ganglion, bipolar and receptor cells, and the radial fibers (Mtiller cells). Two main morphological patterns are significant for the organization of the retina during neurogenesis: a) the cell to cell contacts of migrating cells and b) the spatial arrangement of Mfiller cells which could provide guidelines for migration of neuronal elements.
Developmental Brain Research, 1990
The differentiation of presumptive neural retina following its isolation from rat embryos and growth in explant and monolayer culture has been studied to obtain information regarding the extent to which factors extrinsic and intrinsic to the retina participate in determining molecular and cytological differentiation. Explanted retinal epithelium retained the capacity for mitosis, as shown by [3H]thymidine incorporation, and from the undifferentiated neuroepithelium, retinal cell-types emerged and acquired a laminar organization resembling that in vivo. Characterization of rod photoreceptor cells at both the light and electron microscopic level showed that these cells exhibit differentiated structural features including inner segments, connecting cilia and membranous expansions suggestive of forming outer segments. Immunofluorescent labeling with an antibody to a synaptic vesicle protein, and electron microscopic identification of synaptic elements showed formation of synapses by the photoreceptor cells within the explant. Neurites extending from the explants exhibited growth on laminin, fibronectin and collagen substrates. Since the neurites immunolabeled with antibodies to the 140 kDa subunit of neurofilament and with antibodies to Thy-1, they could be identified as axons of ganglion cells. Antibodies to a variety of cell-type specific antigens showed that the cells expressed molecules associated with the fully differentiated cell. Furthermore, since our approach has been to explant embryonic retina at an age when the antigens are not yet expressed in vivo, the appearance of the antigens in culture represented de novo expression. In contrast, neural retinal cells in dissociated cultures did not exhibit de novo expression of differentiated molecular properties. Collectively, these results indicate that the isolated neural retina is capable of cell birth, commitment and differentiation when the cells are maintained together in explant culture, whereas the environment of monolayer cultures is apparently unable to promote the generation of mature neuronal phenotypes.