Each pigmented epithelial cell bears circumferential actin bundles at its apical level when the pigmented epithelium is established in eyes in situ or in culture in vitro. Welldifferentiated pigmented epithelia in culture were treated with a 50% glycerol solution containing 0.1 M KCI, 5 mM EDTA, and 10 mM sodium phosphate buffer, pH 7.2, for 24 h or more at 4°C. When the glycerinated epithelium was transferred to the ATP solution, each cell constituting the epithelium began to contract . The epithelium was cleaved into many cell groups as a result of contraction of each cell . The periphery of each cell group was lifted to form a cup or vesicle and eventually detached from the substratum . However, those cells that had not adhered tightly and not formed a monolager epithelium with typical polygonal cellular pattern contracted independently as observed in the glycerinated fibroblasts .Contraction of the glycerinated pigmented epithelial cells was inhibited by N-ethylmaleimide but not by cytochalasin B. ITP and UTP also effected the contraction of the glycerinated cells, but GTP and ADP did not. Ca t+ was not required . This contractile model of pigmented epithelium provides a useful experimental system for analyzing the function of actin in cellular morphogenesis.
Singly.dissociated cells from dorsal and ventral iris epithelia (iris iridicu) of adult newts were cultured separately at clonal density to analyse their growth and differentiative capacity. Usually some attached cells began to proliferate on 12th day of culture, and grew with loss of melanosomes to form clonal cell colonies. Up to 30 days after inoculation, most of the clonal colonies formed typical epithelial monolayer sheets which consisted mostly of nonpigmented cells. Then, in some of those colonies, cells piled up together and form typical lens structures containing lens antigens.A month and a half after culturing, 30 to 40% of single iris cells, which had been previously marked, grew to form clonal colonies consisting of more than 100 cells. About 30% of these colonies expressed lens specificity and no significant differences in efficiency of colony formation and differentiation were detected between the dorsal cells and the ventral, suggesting that potent cells capable of transdifferentiating into lens cells are evenly distributed in all parts of the newt iris epithelium.Since G. WOLFF demonstrated experimentally lens regeneration from the iris in the newts (20), the conversion of iris tissue t o lens tissue, now termed transdifferentiation (1 8), has attracted a number of workers in the field of embryology and developmental biology ( 3 , 5, 1 1,13,14,15,17,19,21,22,23), because it is the clearest demonstration that fully differentiated cells can switch from their type of differentiation and be reprogrammed into a different differentiative pathway. Substantial findings obtained through analytical studies of such phenomena might bring about much deeper understanding of cell differentiation in general.In the last few years, the consistent efforts of workers engaged in the problem of lens regeneration have concentrated on establishing much more refined experimental systems t o permit crucial analyses at the cellular and molecular levels (2,4,5, 7,8,10,24,25). EGUCHI and OKADA (8) first demonstrated that the progeny of pigmented retinal cells of chick embryo switched their differentiation into lens cells in clonal culture. We have also demonstrated that cells from iris pigmented epithelia of adult newts transdifferentiated into lens cells in culture (7).One of the most remarkable characteristics of Wolffian regeneration is that a lens is regenerated only from the mid-dorsal margin of iris epithelium after simple lentectomy. Nevertheless, according t o grafting experiments (1 2, 16), the capacity for lens regeneration is highest at the pupillary margin of the mid-dorsal iris; declining both circumferentially around the pupil and proximally away from the pupil toward the neural retina, but there is no lens potency in the ventral iris. ECUCHI and SHINCAI (9) interpreted this situation from the results o f an
The structural and biochemical changes of cytoskeletal components of retinal pigmented epithelial cells were studied during the development of chicken eyes. When the cytoskeletal components of the pigmented epithelial cells from various stages of development were examined by SDS PAGE, actin contents in the cells markedly increased between the 15-d-old and hatching stages. Immunofluorescence microscopy showed that chicken pigmented epithelial cells have two types of actin bundles. One is the circumferential bundle associated with the zonula adherens region as previously reported (Owaribe, K., and H. Masuda, 1982, J. Cell Biol., 95:310-315). The other is the paracrystalline bundle forming the core of the apical projections.The increase in actin contents after the 15-d-old stage is accompanied by the formation and elongation of core filaments of apical projections in the cells. During this period the apical projections extend into extracellular space among outer and inner segments of photoreceptor cells. Accompanying this change is an elongation of the paracrystalline bundles of actin filaments in the core of the projection. By electron microscopy, the bundles decorated with muscle heavy meromyosin showed unidirectional polarity, and had transverse striations with ~12-nm intervals, as determined by optical diffraction of electron micrographs. Since the shape of these bundles was not altered in the presence or absence of Ca 2÷, they seemed not to have villin-like proteins. Unlike the circumferential bundles, the paracrystalline bundles did not contract when exposed to Mg-ATP. These observations indicate that the paracrystalline bundles are structurally and functionally different from the circumferential actin bundles.Actin is a major component of the cytoskeletal and contractile elements in nonmuscle cells. It plays an essential role in cell motility and shape determination of the cell (7,16,20,26,31,34). Depending on the physiological state of the cell, actin undergoes assembly and disassembly, and cellular actin levels are also regulated quantitatively in the cell. Actin content becomes a good marker for cell physiology and morphology in some specialized cells (15,32,35) and transformed cells (17,38).In the present studies, the changes in the cytoskeletal components of retinal pigmented epithelial cells were examined during the development of chicken eyes. Relative contents of a 42-kD protein of the cells from a l-d-old chick were found to be much larger than those of an 11-d-old embryo when 590 assayed by SDS gel electrophoresis. The aim of this study is to determine why there is an increase in a 42-kD protein in the cells and how the cells use this additional protein.The increase in 42-kD protein is found to be closely related to the formation and elongation of apical projections that have paracrystalline core bundles in chicken epithelial cells. MATERIALS AND METHODS Preparation of Pigmented Epithelia:Retinal pigmented epithelia were prepared from chicks of various ages, ranging from 11-d-old embryos ...
The ultrastructure of differentiated colonies developed in vitvo from singly isolated chondrocytes of the sterna of chicken embryos were studied by an improved method for cutting ultra-thin sections for electron microscopy in a plane normal to the plastic substrate. The differentiated colony consisted of a central refractile portion with a metachromatic matrix and a peripheral monolayered ring. Electron microscopic observation of thin sections showed a large central lumen which seems to be filled with cartilaginous matrix and collagen. Its roof is a thin layer of cells, in which the cells come together rather compactly, while the floor of the lumen consists of a monolayer, in which conical cells adhering to the substrate were loosely distributed. Most cells in the central portion are rich in granular endoplasmic reticulum with prominent saccular cisternae and large vacuoles presumably derived from Golgi complexes. The cells adhering to the substrate are actually in contact with it at only a few points across a gap of 40-200A. The rest of the cell surface is free of the substrate.
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