Comparison of Neuronal and Lens Phenotype Expression in the Transdifferentiating Cultures of Nueral Retina with Different Culture Media* (original) (raw)
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Expression of Neuronal Specificities in "Transdifferentiating" Cultures of Neural Retina
Differentiation, 1980
Cells dissociated from the neural retina of embryonic chick differentiate into lens and pigment cells, when cultured in vitro. Using 3.5-day-old and 8.5-day-old chick embryos, we examined whether neuronal specificities would be expressed in such transdifferentiating cultures of neural retinal cells. The synthesis of acetylcholine and gamma-aminobutyric acid (GABA) and the activity of choline acetyl transferase (CAT) was searched for in these cultures. The synthesis of an appreciable amount of these two putative neurotransmitters was detected in cultures of 3.5-day-old embryonic retinas by about 15 days. The activity of CAT was maximum in 7-day cultures of the 3.5-day-old materials and in 2-day cultures of the 8.5-day-old materials, and then decreased. Concomitant with the decrease of CAT-activity, delta-crystallin became detectable and increased thereafter. CAT-activity changed in parallel with the increase in the number of small neuroblast-like cells in cultures. The results demonstrate that the neuronal specificity identified by the appearance of acetylcholine and GABA and of the enzyme for the synthesis of acetylcholine is expressed in the early period of transdifferentiating cultures, which would later differentiate into lens and pigment cells. The possible mechanisms of the transition from neuronal to non-neuroretinal specificities of the transdifferentiating cultures are discussed.
Development, Growth and Differentiation, 1981
Embryonic chick neuroretinal cells transdifferentiate into lens cells during culture in media containing foetal calf serum (F). This process is largely inhibited if horse serum plus supplementary glucose (Hg) is substituted for F. This paper explores the effect of medium changeover (from F to Hg or vice versa) on the subsequent appearance of lens-specific 6-crystallin. If cultures are changed from Hg to F up to 12 days of culture, 6-production at 40 days is similar to that for controls maintained in F throughout. Changeovers between 14 and 17 days progressively inhibit subsequent 6 production, and after 19 days in Hg, lens transdifferentiation cannot be induced by F. Conversely, if cultures are maintained in F for up to 17 days, a changeover to Hg blocks transdifferentiation, whereas similar transfers performed after 19 days give increased 6 production. These results suggest that some retinal cells which will eventually form lens in vitro become so determined between the 12th and 20th days of culture. A mixture of 50% Hg and 50% F medium (FHg) does not support 6 production even after 60 days, but in the absence of supplementary glucose (FH), 6 appears in considerable amounts by 30 days. Both lens and pigment cells are formed extensively during prolonged culture of embryonic neuroretinal (NR) cells in media containing foetal calf serum (
Development, Growth and Differentiation, 1982
Cells dissociated from neural retina of 3.5-day-old chick embryos transdifferentiated extensively into lens cells under the conditions of a cell culture for 3 to 4 weeks. In early satges of cell culture by about 10 days, cultures consisted of small round cells often with cytoplasmic processes(N-cells) and flattened epithelial cells (E-cells). Only N-cells were stained with a fluorescent dye Merocyanine 540. When cells harvested from early cultures were separated into two fractions by centrifugation in Percoll gradient, the specific activity of choline acetyltransferase was much higher in the fraction consisting mainly of N-cells than in other fraction mainly of E-cells. Continuous daily observations as well as cinematographic observations of living cultures indicate that lentoid bodies were often formed in the locations where clusters of N-cells had been found in early stages of culturing. The possibility of transdifferentiation of N-cell clusters into lentoid bodies is discussed. * A part of the present results was reported in a preliminary form (1, 2).
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
Development, Growth and Differentiation, 1982
The accumulation of 6 crystallin (chick lens marker) in cultures of 9 day chick embryo neuroretinal cells is strongly promoted by chick embryo extract (CEE) or foetal calf serum (FCS), but much less so by adult sera (horse, chicken and newborn bovine serum). The "transdifferentiationpromoting" (TP) activity of FCS is absent from dialysed FCS but is largely recovered in the initial dialysis medium (FDM). Similarly, the initial dialysis medium from CEE (EDM) shows strong TP activity, whereas that from chicken or from horse serum does not. We conclude that the proposed TP factor(@ is (are) of relatively low molecular weight. By contrast, horse serum contains macromolecular factor(s) able to inhibit the TP activity of EDM or F D M. Rapid loss of neuronal cells (including those expressing choline acetyltransferase activity) is also observed in media based on FDM, though whether this effect is mediated by the proposed TP factor($ has not been determined. The TP activity is not directly related to growth rate or cell density, since cultures in FDM alone grow poorly yet still accumulate 6 crystallin.
Differentiation and morphogenesis in pellet cultures of developing rat retinal cells
The Journal of Comparative Neurology, 1997
We previously developed a reaggregate cell culture system (pellet cultures) in which retinal neuroepithelial cells proliferate and give rise to rod photoreceptor cells (rods) in vitro (Watanabe and Raff, 1990, Neuron 4:461-467). In the present study, we analyzed cell differentiation and morphogenesis in pellet cultures by using both cell-type-specific markers with immunofluorescence and electron microscopy. We demonstrated that, in addition to rods, the other major retinal cell types, including amacrine cells, bipolar cells, Mü ller cells, and ganglion cells were all present in the pellets, where most were able to develop from dividing precursor cells in vitro. The different cell types in the pellets became organized into two distinct structures: dark rosettes and pale rosettes. The cellular composition of these structures indicated that the dark rosettes correspond to the outer nuclear layer and the pale rosettes to the inner nuclear layer of the normal retina. Ultrastructural studies have indicated that the thin layer of neuronal processes surrounding the dark rosettes correspond to the outer plexiform layer, and the central region of the pale rosettes correspond to the inner plexiform layer of the normal retina. Other features of normal retinal development also occurred in the pellets, including programmed cell death and the formation of inner and outer rod cell segments and synapses. Thus, pellet cultures provide a convenient way to study different aspects of retinal development where one can control the size and the cellular composition of the initial reaggregate.
Tissue culture studies of retinal development
Methods, 2002
Because of a limited number of cell types, a series of well-described cell-type-specific markers and a stereotyped sequence of cell development, the retina has been a valuable model of CNS development. Dissociated and explant cultures have been used to help define some of the requirements for differentiation of each major cell class. In addition to mixed-cell cultures it is now possible to use cell purification or selective growth methods to give cultures of single cell types. Alteration of gene expression by viral infection has proved to be a valuable method to help elucidate developmental pathways.
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.
Developmental Biology, 1982
Chick embryo neuroretinal cells accumulate lens-specific 6-crystallin when cultured in Eagle's minimal essential medium containing 5% foetal calf serum and 5% horse serum (FH), but fail to do so if supplementary glucose is present (FHG). Culture growth rates are similar in the two media, but choline acetyltransferase activity is maintained for longer in FHG medium. By transferring cultures from FHG into FH medium and vice versa, we show that potential lens precursor cells survive for at least 21 days in FHG medium, while some retinal cells become irreversibly committed to crystallin production after 18 days in FH medium.
Experientia, 1997
The outer cornea of larval Xenopus lae6is basophilia), cell elongation, gradual loss of basophilic properties and acquisition of acidophilic properties for can reprogram cell differentiation when cultured in medium conditioned by X. lae6is neural retina crystallin synthesis and accumulation. These events were completely dependent on XRCM or RRCM, (XRCM) or by Rana esculenta neural retina (RRCM). Under these experimental conditions corneal cells suggesting that the neural retina secretes a factor(s) which initiates and sustains lens fibre transdifferentia-showed the same series of cytological changes of fibre cell differentiation observed during ontogenesis and in tion of the corneal epithelial cells. This culture system vivo lens regeneration: enlargement of nuclei and nu-appears to be a suitable one for investigating the concleoli, increase of ribosomal population (cytoplasm trol of lens fibre transdifferentiation in vitro.