Journal o f Neurocytology 8,229-238 (1979)
The formation of gap and tight junctions between retinal pigment cells in cell cultures K. M E L L E R Institut fiir Anatomie, Arbeitsgruppe fiir Experirnentelle Cytologie, Rubr-Universitdt Bocbum, West Germany
Received 6 September 1978; revised 7 December 1978; accepted 13 December 1978
Summary The reformation of the junctional complex of retinal pigment cells was studied after trypsin disaggregation and in vitro reaggregation. Control specimens show a zonula occludens (tight junction) with integrated gap junctions and very large macular gap junctions. Isolation after trypsination results in disaggregation of the large gap junctions and fragmentation of the tight junctions with disaggregation of their integrated gap junctions. After two to four days of incubation the restoration of the zonula occludens is complete. After approximately five days of incubation, large gap junctions are found with a patchy arrangement of particles similar to that seen
in vivo.
Introduction Although many ultrastructural and physiological properties of gap and tight junctions have been elucidated (McNutt and Weinstein, 1973; Staehelin, 1974)we know relatively little about their formation (Yee, 1972; Decker and Friend, 1974; Montesano et al., 1975; Decker, 1976; Elias and Friend, 1976; Gilula et al., 1976). Occluding junctions are found as a complete band around the apical margins of epithelial cells. F arquhar and Palade (1963, 1965) characterized them in thin sections as regions in which the membranes of adjacent cells are so closely apposed that the intercellular space between them is occluded by an apparent fusion of the outer leaflets of the membranes. Electron microscopy of replicas of freeze-etched epithelia shows that the occluding junctions consist of a web of lines of attachment between the cells (Revel et al., 1973; Albertini and Anderson, 1974; Decker and Friend, 1974; Johnson et al., 1974; Decker, 1976). Gap junctions occur as macular or spot-like contacts between the cells and show in freeze-etch specimens a hexagonal array of particles in each of the apposed membranes. In some epithelia both types of cell junction are present in the terminal bar region (McNutt and Weinstein, 1973). In the retinal pigment epithelium a junctional complex has been described (Hudspeth 9 1979 Chapman and Hall Ltd. PHnted in Great Britain
230
MELLER
and Yee, 1973), in which macular gap junctions are integrated in the region occupied by the zonula occludens. These authors demonstrated that pigment epithelium cells are electrically coupled. The ultrastructural observations of Middleton and Pegrum (1976) also show that the dissociated and reaggregated retinal pigment cells of the embryonic chick stabilized junctional contacts after a short time in culture. In a previous publication, we demonstrated the assembly of these junctional complexes in dissociated and reaggregated cells o f the choroid epithelium. The problem of the formation of the zonula occludens and its integrated gap junctions was given special attention (Dermietzel et al., 1977). The aim of the present study is to report on the effects of trypsin isolation on the junctional complex of pigment epithelium cells and to describe the formation of gap and tight junctions during reaggregation of the cells in vitro. Materials and methods
Cell cultures Retinal pigment epithelia of 10-day-old chick embryos were dissected and immersed in a calciumand magnesium-free balanced salt solution (CMF). The pieces of tissue were transferred to 5 ml of preheated (37 ~ C) trypsin medium (0.05% trypsin in CMF). To avoid the sedimentation of the cells on the surface of the glass, the medium was kept in motion by a slowly rotating (1 r/s) magnet stirrer. After 8 min the first dissociated cells were removed and filtered through two-ply silk and placed in a 50 ml centrifuge tube with 20 ml medium (Eagle minimum essential medium, 10% horse serum, 6 mg/ml glucose). Fresh prewarmed trypsin was then added to the pigment material and after 8 min the newly dissociated cells were again collected. This procedure was repeated five times. Light microscopical controls show that more than 90% of the cells are isolated. This disaggregation procedure avoids the mechanical disruption of the tissue, caused, for example, by triturating through a filter or by pipetting. The cell suspension was cultivated for 2-12 days in a rotating Erlenmeyer or in 25 ml Falcon flasks in a CO2-enriched incubator.
Freeze-etching For freeze-etchlng the pigment cell cultures and the pigment epithelia from 6- to 16-day-old embryos were fixed in phosphate-buffered 2.5% glutaraldehyde. After fixation the pigment cells were washed in the same buffer and immersed for variable periods in 30% glycerol-phosphate buffer solution. The specimens were mounted on Balzers' gold disks, rapidly frozen in liquid nitrogen-cooled Freon 22 and stored in liquid nitrogen. Freeze-fracturing and etching was performed in Balzers' BAF 300 apparatus at --115~ C. The replicas were cleaned in sodium hypochlorite, washed in distilled water and mounted on Formvar-coated copper grids. The replicas were examined with a Siemens Elmiskop IA.
Results
Freeze-etching aspects of the control specimens The junctional complexes of control pigment cells were very similar to those described by Hudspeth and Yee (1973) in various species. The morphological differentiation of retinal pigment cells, in the developing chick eye occurs very early
Formation of cell contacts between retinal pigment cells
231
(Meller, 1968), and by the 10th day of incubation these cells possess a well developed junctional complex. As illustrated in Figs. 1 and 2, the junctional complex which surrounds the cellular apices is characterized by the presence of extensive gap junctions and anastomosing ridges of tight junctions. The approximately 9 nm large particles of the gap junctions are arranged in smaller groups or double rows within the gap junctional contacts which vary in size but reach a maximum diameter of 2 ~n~ As pointed out by Hudspeth and Yee (1973), the gap junctions occur only in the region of these junctional complexes and are not present in other regions of the cell surface. Tight junctions extending around the cell periphery form a band with a parallel orientation of strands similar to that found in other epithelia. Several particle aggregates of different sizes are integrated in the network of anastomosing ridges of the zonula occludens. The aspect and extension of the nexus and the zonula occludens do not change qualitatively during the following stages of development (Fig. 3). The extensive gap junctions are situated apical to the zonula occludens.
Disaggregation and in vitro cultures The effects of trypsin during the isolation of pigment cells are summarized in Figs. 4 - 8 . After a short period of trypsination, the large apical gap junctions have lost the typical arrangement of their particles and reveal round patches of variable diameter (Figs. 4 - 6 ) . The surrounding cell membrane areas show particle aggregates that have probably been disaggregated from the gap region. Incomplete trypsination and mechanical disaggregation frequently cause a portion of the membrane of the adjacent cells to remain attached in the gap region. However, when trypsination occurs in several steps as described above, gap junctions are not discernible because they have disaggregated into isolated particles. Trypsination induces a degradation of the zonula occludens and its integrated gap junctions (Figs. 7 and 8). This degradation of the zonula occludens varies from a clear fragmentation of its components to the disorganization of the junctional segments. Immediately after the trypsin procedure for isolation, most cells show only chains of particles (Fig. 7). The sequence of reformation is variable and probably depends on the conditions of the cell culture. However, monolayer and
Figs. 1--3. Retinal pigment epithelium of a 10-day-old chick embryo. Fig. 1. Zonula occludens with integrated nexus (arrow). X 70 000. Fig. 2. A typical large gap junction. • 50 000. Fig. 3. Freeze-fracture of the junctional complex of 16-day-old chick embryo retinal pigment epithelium, x 70 000. Figs. 4--6. Retinal pigment epithelium of a lO-day-old chick embryo. Characteristic pattern of trypsin action during the dissociation procedure. Observe the disorganization of the particle arrangement in gaps before adjacent cells have been dissociated, x 90 000. Figs. 7 and 8. Trypsinized zonula occludens. One can observe isolated chains and groups of particles and fragments of the tight junctions (arrows). x 80 000.
Formation of cell contacts between retinal pigment cells
2 35
suspension cultures differ only slightly in this respect. The most important factor for aggregation to take place is the density of the cultivated cells. A few hours after isolation the pigment cells reaggregate in small clumps in suspension cultures or form islands of cells in flask cultures. The diameter of the clumps increases during the following days, but remains constant after the fourth day of cultivation. In culture flasks, the cells form islands and the number of cells incorporated into the islands increases with prolonged cultivation. These aggregates become flattened and form a monolayer by apposition with neighbouring islands. After two days in vitro, a progressive process of tight junction formation becomes discernible (Figs. 9-12). New fragments or fragments of the former tight junctions are situated in the apical zone of the pigment cells. Chains of particles become visible in close relationship with irregularly-oriented tight junctional strands. These formation processes take place within two to four days. The formation of the nexus begins with the formation of the zonula occludens or usually, the appearance of groups of particles between the newly forming tight junctions. They are not always integrated in all replicas examined during the first week of cultivation but can be found in approximately half of them (Fig. 13). Fig. 14 shows the complete remodelled nexus after trypsination and one week of cultivation. These extensive gap junctions rarely appeared in the cultivated material before the fifth day of incubation. The most significant phenomenon is the reorganization of gap particles in parallel rows giving the macula a mosaic-like appearance. Discussion
Fully developed retinal pigment epithelium cells as shown by Hudspeth and Yee (1973) demonstrate a variety of cell contacts such as zonulae occludentes, zonulae adherentia and especially well-developed gap junctions in the region of the zonula occludens. Physiological studies by these authors demonstrated that these pigment epithelium cells are electrically coupled and that the gap junctions are probably responsible for this coupling. The formation of gap junctional contacts in the early embryonic stage, as reported by other authors (Bennett, 1973, Revel et al., 1973; Revel and Brown, 1976) gives rise to the existence of sites which allow a transmission of metabolically important substances or the passage of factor(s) which might control cell proliferation and differentiation. Ultrastructural studies in reaggregated pigment cells of the retina and the observation of the development of these contacts Figs. 9--12. Retinal pigment epithelium of a 10-day-old chick embryo after trypsination in vitro. Figs. 9 and 10. On the second day of cultivation a remodelling of the zonula occludens becomes evident, x 50 000. Figs. 11 and 12. After four days in vitro an increase in zonulae occludentes with integrated gap junctions can be observed (arrows). x 50 000. Fig. 13. Retinal pigment cells cultured for seven days. During this phase of reaggregation, the junctional complex is composed of relatively continuous strands, x 70 000. Fig. 14. Retinal pigment cells cultured for five days. The arrangement of the particles or the large gap junctions is now similar to that in controls, x 70 000.
236
MELLER
in cultures have been interpreted in connection with the control of locomotion of these cells in vitro (Middleton and Pegrum, 1976). These authors affirmed that these contact specializations develop after the original collisions as the most stable connection between the cells. The in vitro formation of cell contacts has also been demonstrated in other tissues [Pinto da Silva and Gilula, 1972 (fibroblasts); Johnson et al., 1974 (Novikoff hepatoma cells); Dermietzel et al., 1977 (choroid plexus)]. As shown in this study on pigment cells and in a former investigation on the formation of the junctional complex in isolated and reaggregated choroid plexus epithelial cells (Dermietzel et al., 1977), the trypsin effect is variable up to a total disorganization of the junctional structure. The tight junctions break into short segments and the gap junctions disappear almost completely. One principal question remaining concerns the fate of the disaggregated material of the junctional complex. Two possibilities may be considered. (a)This material is disintegrated and degradation is then carried out by lysosomes in an endocytic mode of digestion. This process, observed in our investigation in the choroid plexus (Dermietzel et al., 1977), confirms the observation of Staehelin (1974). However, in the present material a lysosomal degradation of the tight junctions was not observed. (b) Intramembranous particles and the remains of gap and tight junctions will participate in the remodelling of the newly-formed contacts. This assumption cannot be satisfactorily supported by morphological observation only. Decker's observations (1975) indicate that intact protein synthesis may be indispensable for the new formation of contacts, since the addition of drugs such as cycloheximide retards the formation of new contacts between anuran ependymoglial cells. This is concordant with the observations of Moscona and Moscona (1966) that retinal cells exposed to puromycin reaggregate very poorly. The other main unsolved question is the origin of the particles that form gap and tight junctions. The concept of a common particle pool for both kinds of membrane contacts needs verification (Elias and Friend, 1976; Meyer et al., 1977). The mechanism that reorganizes the particles in the membrane also remains unknown. The investigations of Merk and McNutt (1972) on the role of steroid hormones in contact formation and the observations of Sheridan (1976) that dibutyryl cAMP promotes and 8-bromo-cGMP retards gap junction formation show that a diversity of problems and factors is involved in the formation of specific contacts. Gilula (1977) proposed that cell aggregation models would be a promising tool for investigating the relationship of differentiation and formation of cell contacts. The present report shows that the process of formation of cell contacts is reproducible in vitro and therefore a most accurate experimental approach to this problem. Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft (Me 276/9) and the Stiftung Volkswagenwerk (Az 11 2977). I want to acknowledge
F o r m a t i o n o f cell c o n t a c t s b e t w e e n retinal p i g m e n t cells
237
the t e c h n i c a l assistance o f M. Waelsch and R. Maeder. I also t h a n k K. Rascher f o r her help w i t h the English translation and C. Bloch for t y p i n g the m a n u s c r i p t . References ALBERTIN1, D.F. and ANDERSON, E. (1974) The appearance and structure of intercellular connections during the ontogeny of the rabbit ovarian follicle with particular reference to gap junctions. Journal of Cell Biology 63,234--50. BENNETT, M. V. L. (1973) Function of electrotonic junctions in embryonic and adult tissues. Federation Proceedings 32, 65-75. DECKER, R. S. (1975) Influence of thyroxine on anuran gap junctions. Journal of Cell Biology 67, 89a. DECKER, R.S. (1976) Hormonal regulation of gap junction differentiation. Journal of Cell Biology 69, 669-85. DECKER, R. S. and FRIEND, D. S. (1974) Assembly of gap junctions during amphibian neurulation. Journal of Cell Biology 62, 3 2 - 4 7 . DERMIETZEL, R., MELLER, K., TETZLAFF, W. and WAELSCH, M. (1977) In vivo and in vitro
formation of the junctional complex in choroid epithelium. A freeze-etching study. Cell and Tissue Research 181,427--41. ELIAS, P.M. and FRIEND, D.S. (t976) Vitamin-A-induced mucous metaplasia. An in vitro system for modulating tight and gap junction differentiation. Journal of Cell Biology 68, 173--88. FARQUHAR, M.G. and PALADE, G.E. (1963) Junctional complexes in various epithelia. Journal of Cell Biology 17, 375-412. FARQUHAR, M. G. and PALADE, G. E. (1965) Cell junctions in amphibian skin.Journal of Cell Biology 26, 263--91. GILULA, N.B. (1977) Gap junctions and cell communication. In International Cell Biology 1976--1977 (edited by BRINKLEY, B.R. and PORTER, K.R.) pp. 61-69. New York: Rockefeller University Press. GILULA, N. B., FAWCETT, D. W. and AOKI, A. (1976) The Sertoli cell occluding junctions and
gap junctions in mature and developing mammalian testis. Developmental Biology 50, 142--68. HUDSPETH, A.J. and YEE, A.G. (1973) The intercellular junctional complexes of retinal pigment epithelia. Investigative Ophthalmology 12, 354-65. JOHNSON, R., HAMMER, M., SHERIDAN, J. and REVEL, J.-P. (1974)Gap junction formation
between reaggregated Novikoff hepatoma cells. Proceedings of the National Academy of Sciences, USA 71, 4536-40. McNUTT, N.S. and WEINSTEIN, R.S. (1973) Membrane ultrastructure at mammalian intercellular junctions. Progress in Biophysics and Molecular Biology 26, 47-101. MELLER, K. (1968) Histo- und Zytogenese der sicb entwickelnden Retina. Eine elektronenmikroskopiscbe Studie. Stuttgart: Gustav Fischer. MERK, F.B. and McNUTT, N.S. (1972) Nexus junctions between dividing and interphase granulosa cells of rat ovary. Journal of Cell Biology 55, 511--9. MEYER, R., POSALAKY, Z. and McGINLEY, D. (1977) Intercellular junction development in
maturing rat seminiferous tubules. Journal of Ultrastructure Research 6 1 , 2 7 1 - 8 3 . MIDDLETON, C. A. and PEGRUM, S. M. (1976) Contacts between pigmented retina epithelial
cells in culture. Journal of Cell Science 22,371-83. MONTESANO, R., FRIEND, D. S., PERRELET, A. and ORCI, L. (1975) In vivo assembly of tight
junctions in fetal rat liver. Journal of Cell Biology 67, 310--9. MOSCONA, H.M. and MOSCONA, A.A. (1966) Inhibition of cell aggregation in vitro by puromycin. Experimental Cell Research 41,703--6.
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PINTO DA SILVA, P. and GILULA, N.B. (1972) Gap junctions in normal and transformed fibroblasts in culture. Experimenlal Cell Researcb 7 1 , 3 9 3 - 4 0 1 . REVEL, J.-P., VlP, V. and CHANG, L. L. (1973) Cell junctions in the early chick embryo - a freeze etch study. Developmental Biology 3 5 , 3 0 2 - 1 7 . REVEL, J.-P. and BROWN, S. (1976) Cell junctions in development, with particular reference to the neural tube. Cold Spring Harbor Symposia on Quantitative Biology 4 0 , 4 4 3 - 5 5 . s HE RI DAN, J. D. (1976) Cell coupling and cell communication during embryogenesis. In Tbe Cell Surface in Animal Embryogenesis and Development. Vol. 1. (edited by POSTE, G. and NICOLSON, G. L.) pp. 409--447. Amsterdam: Elsevier/North Holland. STAEHELIN, L. A. (1974) Structure and function of intercellular junctions. International Review of Cytology 39, 191--283. YEE, A. G. (1972) Gap junctions between hepatocytes in regenerating rat liver. Journal of Cell Biology 55,294a.
The formation of gap and tight junctions between retinal pigment cells in cell cultures K. M E L L E R Institut fiir Anatomie, Arbeitsgruppe fiir Experirnentelle Cytologie, Rubr-Universitdt Bocbum, West Germany
Received 6 September 1978; revised 7 December 1978; accepted 13 December 1978
Summary The reformation of the junctional complex of retinal pigment cells was studied after trypsin disaggregation and in vitro reaggregation. Control specimens show a zonula occludens (tight junction) with integrated gap junctions and very large macular gap junctions. Isolation after trypsination results in disaggregation of the large gap junctions and fragmentation of the tight junctions with disaggregation of their integrated gap junctions. After two to four days of incubation the restoration of the zonula occludens is complete. After approximately five days of incubation, large gap junctions are found with a patchy arrangement of particles similar to that seen
in vivo.
Introduction Although many ultrastructural and physiological properties of gap and tight junctions have been elucidated (McNutt and Weinstein, 1973; Staehelin, 1974)we know relatively little about their formation (Yee, 1972; Decker and Friend, 1974; Montesano et al., 1975; Decker, 1976; Elias and Friend, 1976; Gilula et al., 1976). Occluding junctions are found as a complete band around the apical margins of epithelial cells. F arquhar and Palade (1963, 1965) characterized them in thin sections as regions in which the membranes of adjacent cells are so closely apposed that the intercellular space between them is occluded by an apparent fusion of the outer leaflets of the membranes. Electron microscopy of replicas of freeze-etched epithelia shows that the occluding junctions consist of a web of lines of attachment between the cells (Revel et al., 1973; Albertini and Anderson, 1974; Decker and Friend, 1974; Johnson et al., 1974; Decker, 1976). Gap junctions occur as macular or spot-like contacts between the cells and show in freeze-etch specimens a hexagonal array of particles in each of the apposed membranes. In some epithelia both types of cell junction are present in the terminal bar region (McNutt and Weinstein, 1973). In the retinal pigment epithelium a junctional complex has been described (Hudspeth 9 1979 Chapman and Hall Ltd. PHnted in Great Britain
230
MELLER
and Yee, 1973), in which macular gap junctions are integrated in the region occupied by the zonula occludens. These authors demonstrated that pigment epithelium cells are electrically coupled. The ultrastructural observations of Middleton and Pegrum (1976) also show that the dissociated and reaggregated retinal pigment cells of the embryonic chick stabilized junctional contacts after a short time in culture. In a previous publication, we demonstrated the assembly of these junctional complexes in dissociated and reaggregated cells o f the choroid epithelium. The problem of the formation of the zonula occludens and its integrated gap junctions was given special attention (Dermietzel et al., 1977). The aim of the present study is to report on the effects of trypsin isolation on the junctional complex of pigment epithelium cells and to describe the formation of gap and tight junctions during reaggregation of the cells in vitro. Materials and methods
Cell cultures Retinal pigment epithelia of 10-day-old chick embryos were dissected and immersed in a calciumand magnesium-free balanced salt solution (CMF). The pieces of tissue were transferred to 5 ml of preheated (37 ~ C) trypsin medium (0.05% trypsin in CMF). To avoid the sedimentation of the cells on the surface of the glass, the medium was kept in motion by a slowly rotating (1 r/s) magnet stirrer. After 8 min the first dissociated cells were removed and filtered through two-ply silk and placed in a 50 ml centrifuge tube with 20 ml medium (Eagle minimum essential medium, 10% horse serum, 6 mg/ml glucose). Fresh prewarmed trypsin was then added to the pigment material and after 8 min the newly dissociated cells were again collected. This procedure was repeated five times. Light microscopical controls show that more than 90% of the cells are isolated. This disaggregation procedure avoids the mechanical disruption of the tissue, caused, for example, by triturating through a filter or by pipetting. The cell suspension was cultivated for 2-12 days in a rotating Erlenmeyer or in 25 ml Falcon flasks in a CO2-enriched incubator.
Freeze-etching For freeze-etchlng the pigment cell cultures and the pigment epithelia from 6- to 16-day-old embryos were fixed in phosphate-buffered 2.5% glutaraldehyde. After fixation the pigment cells were washed in the same buffer and immersed for variable periods in 30% glycerol-phosphate buffer solution. The specimens were mounted on Balzers' gold disks, rapidly frozen in liquid nitrogen-cooled Freon 22 and stored in liquid nitrogen. Freeze-fracturing and etching was performed in Balzers' BAF 300 apparatus at --115~ C. The replicas were cleaned in sodium hypochlorite, washed in distilled water and mounted on Formvar-coated copper grids. The replicas were examined with a Siemens Elmiskop IA.
Results
Freeze-etching aspects of the control specimens The junctional complexes of control pigment cells were very similar to those described by Hudspeth and Yee (1973) in various species. The morphological differentiation of retinal pigment cells, in the developing chick eye occurs very early
Formation of cell contacts between retinal pigment cells
231
(Meller, 1968), and by the 10th day of incubation these cells possess a well developed junctional complex. As illustrated in Figs. 1 and 2, the junctional complex which surrounds the cellular apices is characterized by the presence of extensive gap junctions and anastomosing ridges of tight junctions. The approximately 9 nm large particles of the gap junctions are arranged in smaller groups or double rows within the gap junctional contacts which vary in size but reach a maximum diameter of 2 ~n~ As pointed out by Hudspeth and Yee (1973), the gap junctions occur only in the region of these junctional complexes and are not present in other regions of the cell surface. Tight junctions extending around the cell periphery form a band with a parallel orientation of strands similar to that found in other epithelia. Several particle aggregates of different sizes are integrated in the network of anastomosing ridges of the zonula occludens. The aspect and extension of the nexus and the zonula occludens do not change qualitatively during the following stages of development (Fig. 3). The extensive gap junctions are situated apical to the zonula occludens.
Disaggregation and in vitro cultures The effects of trypsin during the isolation of pigment cells are summarized in Figs. 4 - 8 . After a short period of trypsination, the large apical gap junctions have lost the typical arrangement of their particles and reveal round patches of variable diameter (Figs. 4 - 6 ) . The surrounding cell membrane areas show particle aggregates that have probably been disaggregated from the gap region. Incomplete trypsination and mechanical disaggregation frequently cause a portion of the membrane of the adjacent cells to remain attached in the gap region. However, when trypsination occurs in several steps as described above, gap junctions are not discernible because they have disaggregated into isolated particles. Trypsination induces a degradation of the zonula occludens and its integrated gap junctions (Figs. 7 and 8). This degradation of the zonula occludens varies from a clear fragmentation of its components to the disorganization of the junctional segments. Immediately after the trypsin procedure for isolation, most cells show only chains of particles (Fig. 7). The sequence of reformation is variable and probably depends on the conditions of the cell culture. However, monolayer and
Figs. 1--3. Retinal pigment epithelium of a 10-day-old chick embryo. Fig. 1. Zonula occludens with integrated nexus (arrow). X 70 000. Fig. 2. A typical large gap junction. • 50 000. Fig. 3. Freeze-fracture of the junctional complex of 16-day-old chick embryo retinal pigment epithelium, x 70 000. Figs. 4--6. Retinal pigment epithelium of a lO-day-old chick embryo. Characteristic pattern of trypsin action during the dissociation procedure. Observe the disorganization of the particle arrangement in gaps before adjacent cells have been dissociated, x 90 000. Figs. 7 and 8. Trypsinized zonula occludens. One can observe isolated chains and groups of particles and fragments of the tight junctions (arrows). x 80 000.
Formation of cell contacts between retinal pigment cells
2 35
suspension cultures differ only slightly in this respect. The most important factor for aggregation to take place is the density of the cultivated cells. A few hours after isolation the pigment cells reaggregate in small clumps in suspension cultures or form islands of cells in flask cultures. The diameter of the clumps increases during the following days, but remains constant after the fourth day of cultivation. In culture flasks, the cells form islands and the number of cells incorporated into the islands increases with prolonged cultivation. These aggregates become flattened and form a monolayer by apposition with neighbouring islands. After two days in vitro, a progressive process of tight junction formation becomes discernible (Figs. 9-12). New fragments or fragments of the former tight junctions are situated in the apical zone of the pigment cells. Chains of particles become visible in close relationship with irregularly-oriented tight junctional strands. These formation processes take place within two to four days. The formation of the nexus begins with the formation of the zonula occludens or usually, the appearance of groups of particles between the newly forming tight junctions. They are not always integrated in all replicas examined during the first week of cultivation but can be found in approximately half of them (Fig. 13). Fig. 14 shows the complete remodelled nexus after trypsination and one week of cultivation. These extensive gap junctions rarely appeared in the cultivated material before the fifth day of incubation. The most significant phenomenon is the reorganization of gap particles in parallel rows giving the macula a mosaic-like appearance. Discussion
Fully developed retinal pigment epithelium cells as shown by Hudspeth and Yee (1973) demonstrate a variety of cell contacts such as zonulae occludentes, zonulae adherentia and especially well-developed gap junctions in the region of the zonula occludens. Physiological studies by these authors demonstrated that these pigment epithelium cells are electrically coupled and that the gap junctions are probably responsible for this coupling. The formation of gap junctional contacts in the early embryonic stage, as reported by other authors (Bennett, 1973, Revel et al., 1973; Revel and Brown, 1976) gives rise to the existence of sites which allow a transmission of metabolically important substances or the passage of factor(s) which might control cell proliferation and differentiation. Ultrastructural studies in reaggregated pigment cells of the retina and the observation of the development of these contacts Figs. 9--12. Retinal pigment epithelium of a 10-day-old chick embryo after trypsination in vitro. Figs. 9 and 10. On the second day of cultivation a remodelling of the zonula occludens becomes evident, x 50 000. Figs. 11 and 12. After four days in vitro an increase in zonulae occludentes with integrated gap junctions can be observed (arrows). x 50 000. Fig. 13. Retinal pigment cells cultured for seven days. During this phase of reaggregation, the junctional complex is composed of relatively continuous strands, x 70 000. Fig. 14. Retinal pigment cells cultured for five days. The arrangement of the particles or the large gap junctions is now similar to that in controls, x 70 000.
236
MELLER
in cultures have been interpreted in connection with the control of locomotion of these cells in vitro (Middleton and Pegrum, 1976). These authors affirmed that these contact specializations develop after the original collisions as the most stable connection between the cells. The in vitro formation of cell contacts has also been demonstrated in other tissues [Pinto da Silva and Gilula, 1972 (fibroblasts); Johnson et al., 1974 (Novikoff hepatoma cells); Dermietzel et al., 1977 (choroid plexus)]. As shown in this study on pigment cells and in a former investigation on the formation of the junctional complex in isolated and reaggregated choroid plexus epithelial cells (Dermietzel et al., 1977), the trypsin effect is variable up to a total disorganization of the junctional structure. The tight junctions break into short segments and the gap junctions disappear almost completely. One principal question remaining concerns the fate of the disaggregated material of the junctional complex. Two possibilities may be considered. (a)This material is disintegrated and degradation is then carried out by lysosomes in an endocytic mode of digestion. This process, observed in our investigation in the choroid plexus (Dermietzel et al., 1977), confirms the observation of Staehelin (1974). However, in the present material a lysosomal degradation of the tight junctions was not observed. (b) Intramembranous particles and the remains of gap and tight junctions will participate in the remodelling of the newly-formed contacts. This assumption cannot be satisfactorily supported by morphological observation only. Decker's observations (1975) indicate that intact protein synthesis may be indispensable for the new formation of contacts, since the addition of drugs such as cycloheximide retards the formation of new contacts between anuran ependymoglial cells. This is concordant with the observations of Moscona and Moscona (1966) that retinal cells exposed to puromycin reaggregate very poorly. The other main unsolved question is the origin of the particles that form gap and tight junctions. The concept of a common particle pool for both kinds of membrane contacts needs verification (Elias and Friend, 1976; Meyer et al., 1977). The mechanism that reorganizes the particles in the membrane also remains unknown. The investigations of Merk and McNutt (1972) on the role of steroid hormones in contact formation and the observations of Sheridan (1976) that dibutyryl cAMP promotes and 8-bromo-cGMP retards gap junction formation show that a diversity of problems and factors is involved in the formation of specific contacts. Gilula (1977) proposed that cell aggregation models would be a promising tool for investigating the relationship of differentiation and formation of cell contacts. The present report shows that the process of formation of cell contacts is reproducible in vitro and therefore a most accurate experimental approach to this problem. Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft (Me 276/9) and the Stiftung Volkswagenwerk (Az 11 2977). I want to acknowledge
F o r m a t i o n o f cell c o n t a c t s b e t w e e n retinal p i g m e n t cells
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the t e c h n i c a l assistance o f M. Waelsch and R. Maeder. I also t h a n k K. Rascher f o r her help w i t h the English translation and C. Bloch for t y p i n g the m a n u s c r i p t . References ALBERTIN1, D.F. and ANDERSON, E. (1974) The appearance and structure of intercellular connections during the ontogeny of the rabbit ovarian follicle with particular reference to gap junctions. Journal of Cell Biology 63,234--50. BENNETT, M. V. L. (1973) Function of electrotonic junctions in embryonic and adult tissues. Federation Proceedings 32, 65-75. DECKER, R. S. (1975) Influence of thyroxine on anuran gap junctions. Journal of Cell Biology 67, 89a. DECKER, R.S. (1976) Hormonal regulation of gap junction differentiation. Journal of Cell Biology 69, 669-85. DECKER, R. S. and FRIEND, D. S. (1974) Assembly of gap junctions during amphibian neurulation. Journal of Cell Biology 62, 3 2 - 4 7 . DERMIETZEL, R., MELLER, K., TETZLAFF, W. and WAELSCH, M. (1977) In vivo and in vitro
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