Primed in Sweden Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any formreserved ISSN W144827
Experimental Cell Research 105 (1977) 3 13-323
RESPONSE CELLS
OF EPITHELIAL TO CULTURE
OBSERVED
AND
MESENCHYMAL
ON BASEMENT
BY SCANNING
LAMELLA
MICROSCOPY
JANE OVERTON Whitman Laboratory,
University of Chicago, Chicago, IL 60637, USA
SUMMARY The basement lamella of Xenopus tadpole skin has been viewed in situ by scanning microscopy, then isolated by trypsin treatment and used as a substrate for cell culture. The basal lamina may also be viewed after EDTA treatment. Responses of epithelial and mesenchymal cells to the lamella have been compared. Mesenchymal cells from chick skin and heart ventricle flatten and attach between the plies of the lamella, then infiltrate it. Myoblasts appear to move less readily within the lamella. Embryonic Xenopus skin epithelium spreads over the surface. Isolated chick skin epithelial cells first begin to spread, then round up and eventually attach to each other in clusters which form a flat basal surface above the lamella. Thus epithelial and mesenchymal cells cultured on this isolated extracellular material mimic aspects of normal tissue organization.
There is increasing interest in the role of the extracellular matrix in development [ 10, 13, 231. However, there are relatively few studies such as those of Meier & Hay [14], Slavkin et al. [22] or Reddi & Anderson [21] in which cell-free matrix material in its original conformation has been recombined with living cells. In the present work, embryonic epithelial or mesenchymal cells have been cultured on the basement lamella of amphibian larval skin in order to assay differences in response to this many layered orthogonal array of collagen fibers. The behavior of cells as indicated by cell shape, alignment or distribution has been followed in an attempt to distinguish differences that may not be apparent under more conventional tissue culture conditions. Collagen fiber systems have been isolated from embryonic chick skin [19], chick duodenum [24] and larval amphibian skin
[18] by a heavy trypsin treatment which removes cellular tissue components, after which the fiber conformation may be viewed by scanning microscopy. This procedure appears to leave unaltered the main course of fiber direction. When the technique is applied to the tadpole the entire basement lamella is exposed [18]. Collagen fibers in these cell-free preparations maintain their orthogonal arrangement. The only apparent divergence from the spatial arrangement found in intact tissue is a variable degree of lateral adhesion between adjacent parallel fibers. The flat lateral surface of the tadpole tail serves as a convenient substrate for cell culture. MATERIALS
AND METHODS
Tadpoles of Xenopus laevis were raised in the laboratory [17] to stages 4%52 when their tails were excised and used as a source of basement lamella. For isolation Exp
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Fig. 1. Intact Xenopus tail which has been dissected after drying. E, epidermis; L, basement lamella; I, basal lamina. x 3 400.
Fig.
of the lamella bv EDTA treatment tails were rinsed briefly in penicillin-streptomycin mixture (Microbiological Associates, 5000 U/ml), then incubated 16 h in calcium-magnesium free Hanks’ solution (CMF) containing 0.5 % EDTA. The epithelium could then be stripped off either with forceps or by a 3 min treatment in an ultrasonic cleaning apparatus (L-2R 320 Ultrasonic). Cell-free preparations- were obtained in some cases by this method, but results were not sufficiently reliable to be used for extensive experiments, so this approach was abandoned. For treatment with trypsin, tails were fixed 30 min or more in 70% alcohol, brought gradually to water, then incubated 30 min in 0.1% trypsin (I : 250, GIBCO) in Hanks’ solution adjusted to pH 7.8 and finally rinsed in water. This procedure routinely produced cell-free lamellae. After lamellae were exposed, tails were placed in culture medium on a glass slide in a 35 mm Petri dish. They were positioned with the right side upwards and held in place with glass bridges. For culture of amphibian tissues, the axial region of stage 26 embryos was excised and explanted onto the tail in culture medium consisting of Niu-Twitty solution. For culture of chick skin, tissue was removed from the back of 7 dav embrvos. incubated 6 min at 4°C in 2 % trypsin (1 : 250) in Hanks’ solution after which the eoithelium could be lifted off. Separated eithelial and mesenchymal components were dissociated by incuba-
tion in 0.1% trypsin and 0.5 % EDTA (1 : 250) in CMF for 30 min at 37°C. After rinsing 3 times in Hanks’ solution. cells were disoersed in culture medium bv gentle pipetting. The dispersed cells were then pipette8 onto the positioned tail. For culture of 8 or 9 day chick heart ver&cle cells, excised tissue was dissociated in the same wav as skin. Culture medium for chick cells consisted ofieibovitz L-15 medium (GIBCO) with 0.5 mgl ml L-elutamine. 10% fetal calf serum (GIBCO), 50 Ul ml each of penicillin and streptomycin’, and 0. i mg/ml deoxvribonuclease (Worthinnton). After culture, preparations were fixed in Kamovskv’s fixative fill. followed bv nost-fixation in 1% osmium tetroxidd’ in collidineV buffer. Preparations were then dehydrated in a series of alcohols and amyl acetate, dried in a Tousimis critical point apparatus using CO, as the transitional fluid and coated with gold-palladium in a Hummer gold vapor coater. Preparations were viewed with an ETEC Autoscan. For observation of the basement lamella and basal lamina in situ, intact tails were fixed, dehydrated and dried as described above, then dissected with a fine tungsten needle before coating. For transmission microscopy, tissue fixed in Karnovsky’s fixative and osmium, as described above, was dehydrated in a series of alcohols and propylene oxide, embedded in Araldite, sectioned with adiamond knife on a Porter-Blum MT2 ultramicrotome and viewed with a Hitachi HU-l1A electron microscope.
Exp Cell
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2. Higher magnification of area indicated by the arrow in fig. I. X20000.
Culture
Fig. 3. Denuded surface of Xenopus tail after EDTA treatment. X20000. Fig. 4. Epithelial outgrowth fromXenopus embryo explant onto tail denuded with EDTA. x6000.
RESULTS The substrate
The basement lamella viewed in situ (figs 1 and 2) shovirs a very regular spacing of orthogonally disposed layers of collagen fibers. Some fibers have been disarranged as a result of dissection. The collagen fibers appear to adhere tightly to the basal lamina (fig. 2). The basal lamina has a textured appearance, but no regular substructure has been resolved.
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5. Denuded surface of Xenopus tail after trypsin treatment. X20000. Fig. 6. Epithelial outgrowth fromXenopus embryo explant onto tail denuded with trypsin. x 10000. Fig.
Methods for separating epithelial cells from tissue have shown that with use of EDTA the basal lamina is left behind [lo] whereas it is removed with trypsin [2,4]. In the present experiments, EDTA treatment left a rough surface (figs 3 and 4) with occasional gaps through which the underlying collagen fibers of the lamella could be observed (fig. 4). Viewed at a higher magnification (fig. 3), the lamina shows many small pits, and is obviously imperfectly Exp Cdl
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preserved by the methods used here. Yet a comparison of this surface with the lamina as seen in situ (fig. 2) indicates that a substantial tissue component remains. After trypsin treatment the orthogonal array of collagen fibers can be clearly seen and four or more plies can be distinguished when viewing from the external surface (fig. 5). Individual collagen fibers tend to adhere laterally forming groups of as many as 8 or 10 in some cases. This clustering occurs after trypsin treatment and seems to be present to about the same degree after critical point drying as judged by inspection of sectioned material with transmission microscopy [18]. As a result, the spaces between fibers to which a cultured cell on this surface is exposed may be roughly 200 mm. Observation by transmission microscopy of sectioned trypsinized tails indicates that cells have been removed from the inner region of the tail as well as from the surface. Pigment granules of the chromatophores remain and serve as a convenient marker of the proximal surface of the lamella. Xenopus embryonic epithelium Embryonic axial regions grown as explants on basal lamina or basement lamella as prepared here send out epithelial sheets which extend over the surface of the substrate. At the free edge of such outgrowths on the lamina, flattened lamellipodia bear processes which show no particular relation to the collagen arrangement which lies beneath (fig. 4). On the lamella, cell processes tend to follow the direction of the superficial collagen fibers (fig. 6). No ruffling was seen. In both instances considerable areas of the surface may be covered with cells. Because of the difficulty encountered in preparing clean surfaces of larnina, other cell types were grown only on the lamella. Exp Cell
Res IOS (1977)
Chick skin Epithelial and mesenchymal skin components were detached and cultured separately as dispersed cells on the lamella. By 2 h mesenchymal cells had flattened on the surface and typically had sent out a number of broad cell processes which were buried beneath the collagen at their terminal regions (fig. 7). By 4 h (fig. 8) cells were spread extensively and often buried deeper. Cellular extensions tended to be closely aligned with lower plies of collagen. A cross-section of the lamella viewed by transmission microscopy at 16 h shows mesenchyme cells layered between the plies (fig. 9). Although the collagen maintains its orthogonal arrangement over this period in some regions, in the vicinity of cells it tends to become disorganized (cf figs 8 and 10). Viewing such a preparation from the surface, it is evident that many cells have moved into the interior of the lamella. Cells not only move into but through the lamella (fig. 9). They are commonly found by 16 h in the interior of the fin. In all the stages of cell spreading and dispersion which were examined, cell bodies and cell processes were characteristically flattened with no evidence of blebbing or ruffling. Many epidermal cells have begun to spread after 2 h of culture on the lamella and their processes tend to align with the collagen fibers (fig. 11). These cells can be compared with cells in the same culture which have settled on the coverslip rather than on the surface of the fin. The cells on glass are somewhat more flattened. By 4 h, cells on glass have continued to flatten, while those on the lamella, in contrast, have become more rounded (fig. 12). Cell processes are now largely or completely withdrawn, but points of attachment to the collagen fibers persist and fiber organization in
Culture on basement lamella
Fig. 7. Chick skin mesenchyme cell on basement lamella, 2 h. x4000. Fig. 8. Mesenchyme cell on basement lamella, 4 h. x2ooo. Fig. 9. Thin section showing mesenchyme cells within pigand beneath the basement lamella. I6 h. Arrow, 21-771815
317
ment granules from a Xenopus chromatophore which mark the inner surface of the basement lamella. X15WO. Fig. IO. Mesenchyme cells within the basement lamella, 6 h. x2000.
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the immediate vicinity of the cells is disturbed. These compact cells characteristically have one or more large rounded blebs. By 16 h (figs 13 and 14) cells are usually found in clusters at the surface. Although the orthogonality of the lamella is maintained at a distance from these clusters, in regions near the cells, the pattern is distorted. In cross-sections of the lamella viewed by transmission microscopy (fig. 13) the basal surface of the clusters is seen as smooth and flattened, resting above the collagen. Nuclei are elongated and lie close together, and the cells of this reconstructed epithelial fragment have relatively little surface exposed to the collagen. Beneath these clusters, the collagen is always distorted and frequently lies in accordion-like pleats. A suggestion of such an arrangement is seen in fig. 13. No evidence of a basal lamina was seen beneath these epithelial clusters, but the longest culture period was 16 h, and observations of others [3, 41 indicate that l-2 days are usually required for reformation of the lamina. Chick heart ventricle The heart ventricle contains cells of a number of types but myoblasts and fibroblasts on which attention is focused here are in the majority. After 2 h of culture on the lamella, suspensions of heart cells had given rise to cells of two characteristic conformations (fig. 15) interpreted here as myoblasts and libroblasts. Fibroblasts had begun to flatten and spread by sending out a number of broad processes which had been inserted beneath the upper ply of collagen and tended to be aligned with lower plies. Their conformation and behavior was similar to skin libroblasts throughout the period studied, They could be seen at 16 h within the lamella, often aligned between plies, and by this time in numerous instances they Exp CeNRes
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were found beneath the lamella as well. Myoblasts which could be identified with certainty by transmission microscopy when contractile elements were seen, showed a very different behavior. They tended to adhere near the surface of the lamella as judged by a transmission microscopy study of sectioned material. Bipolar cells which were rounded in cross-section, seen at 2 h (figs 15 and 16), could also be seen near the surface at 16 h (figs 17 and 18). At 2 h these bipolar cells showed a very strict alignment with the substrate. Orientation is clearly parallel to the second ply and at right angles to the first. Frequently the majority of the contractile elements in the cytoplasm of a myoblast ran parallel to nearby collagen bundles (fig. 17). The orthogonality of the lamella was distorted by 16 h, perhaps due to contractions of myoblasts as well as activity of fibroblasts (cf figs 16 and 18). Thus, though these cells may send processes deep into the collagen and become covered by collagen fibers, they do not appear to move as freely within and beneath the lamella as do fibroblasts.
DISCUSSION Early attempts to disaggregate cells employing trypsin [26] led to the development of a technique for dissociating and reassociating cells from tissues of higher vertebrates [ 161 which has become a widely used method for analysis of tissue organization. In the present study, cellular and non-cellular tissue components are trypsin dissociated and recombined. Just as reassociated cells give rise to normal tissue patterns, these experiments with cellular and mimic non-cellular tissue components aspects of normal tissue organization; epithelial cells form a smooth basal surface
Culture on basement lamella
II. Chick skin epithelial cell on basement lamella, 2 h. x5000. Fig. 12. Skin epithelial cells on basement lamella, 4 h.
Fig.
x2ooo.
3 19
13. Thin section of skin epithelial cells resting on top of basement lamella. ~4 500. Fig. 14. Cluster of skin epithelial cells on outer surface of basement lamella. x I 500.
Fig.
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Fig. IS. Cells from chick heart ventricle on basement lamella, 2 h. E, presumed fibroblast. M, presumed myoblast. x2 300. Fig. 16. Bipolar ventricle cell showing strict alignment with fibers of basement lamella, 2 h. X2 500.
Exp Cell
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Fig. 17. surface Fig. 18. at 16 h.
Myoblasts seen in thin section lying near the of the basement lamella, 16 h. X 14000. Presumed myoblasts near the lamella surface x4000.
Culture
above the collagen and mesenchymal cells invade and penetrate it. Epithelial cells of the Xenopus embryo grow across the surface of the basement lamella in a sheet the leading edge of which sends out processes which show a tendency to align with collagen fibers of the lamella. Likewise, trypsin dissociated epithelial cells of the chick skin, early in culture, show a tendency to flatten on the surface and send out microspikes which frequently follow the direction of adjacent fibers. These initially flattened cells then round up, and by 4 h in culture show a number of large hemispherical blebs. In contrast, cells in these same cultures which have settled on the glass coverslip continue to flatten. By 16 h cells on the lamella which have made contact with each other are associated in clusters. Scanning images suggest that the initially flattened cells make contact with the collagen fibers and move about, distorting the orthogonal pattern. As contact between cells in clusters increases, reducing the cell surface facing the collagen, the lamella appears to become compressed forming irregular pleats. It is possible that the distortion of the normal lamellar pattern is caused not only by physical forces exerted by the cells but also by digestion, but there is no clear evidence for this. The behavior of these epithelial cells is reminiscent of reports in the literature that isolated epithelial cells, as opposed to epithelial cells in sheets, have a tendency to round up. Observation of living cultures of chick skin, intestinal and cornea1 epithelial sheets grown on glass indicate that cells which leave a spreading sheet round up and bleb [5, 61. Also, chick pigmented retina epithelium cells dissociated and plated out on reconstituted collagen are poorly spread and show much blebbing early in the life of the culture [15]. This tendency to round up
on basement
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presumably is one factor which contributes to keeping skin epithelial cells on the surface of the lamella. Why the clustered cells in the present experiments fail to spread is not clear. Possibly the weakened and distorted organization of the collagen in the vicinity of the clusters will not support spreading. At least one factor which controls the distribution of these epithelial cells on the lamella appears to be the strong tendency for the cells to adhere to each other [5]. Mesenchymal cells in all cases, skin fibroblasts, heart fibroblasts and heart myoblasts, always showed a tendency to penetrate the lamella. These cells all have flattened extensions and in the case of fibroblasts, the cell bodies are also extremely flattened. Cell processes show a pronounced orientation in accordance with the organization of the substrate. Cell processes, and eventually whole cells, move between the plies of the basement lamella and fibroblasts may even move through it to the proximal side. In the living amphibian, mesenchymal cells of the skin also penetrate the basement lamella of the tail at metamorphosis. Fine structure studies of the process in normal metamorphosis [8] and in hormone-treated tadpoles [25] show that the invading cells may be layered between the plies as in the case described here. At metamorphosis the basement lamella becomes increasingly loosened and frayed, and the invading cells phagocytize large amounts of collagen, There was no evidence in the present study that collagen was phagocytized. It is possible, as in the case of epithelial cells, that it is broken down enzymatically. In any event, its organization is considerably distorted by the end of 16 h of culture in the presence of these cells. With each cell type studied here there Exp Cdl
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was evidence that cell processes were guided by the orientation of the substrate. This aspect of cell behavior has been recognized for a long time [9]. However, the various cell types showed great variability in the degree of response they exhibited. The most striking orientation was seen in myoblasts where a bipolar cell body was oriented parallel to the second ply and at right angles to the first. Cardiac myoblasts grown on a collagen gel are not necessarily bipolar [12], but these cells do exhibit the greatest polarity of those used in this study. Highly polarized cells showed the most orientation and possibly, the conformation of the basement lamella contributed to the extreme degree of polarization [27]. Polarization of a cell or its capacity to become polarized may be correlated with its capacity to infiltrate the lamella. But this is not the only important factor, since fibroblasts which tended to be less polarized infiltrated the lamella more extensively than myoblasts. Ruffling of the cell surface which is a common type of surface movement of cells in culture, was almost completely absent under the culture conditions used here. This is consistent with the observations of Bard & Hay [I] that cells moving through their normal collagen matrix do not exhibit this surface behavior. DiPasquale [5] has compared the behavior of embryonic epithelial and mesenchymal cells and points out that in both cases lamellipods, ruffles and blebs are formed, although to different extents. The kinds of behavioral differences studied in the present work will presumably be clarified in part by further studies of plasma membrane glycoprotein which mediates adhesion of fibroblasts to collagen [20, 281. The culture of cells on collagen components isolated from tissue as in this study not only shows a very different response in E.rp Cd
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epithelial and mesenchymal cells, but elicits behavior different from that seen when cells are grown on artificial substrates. For example, epithelial cells grown on filters will typically send fine cell processes deep into the underlying cavities. The method of isolating the basement lamella with trypsin which was used here obviously left a structure depleted of some components. In addition, the lamella was distorted since collagen fibers which are normally evenly spaced were sometimes clumped to give intervening spaces of -200 nm. Furthermore, the basal lamina had been removed. It is possible that isolation of this intercellular region in a more intact state would evoke a different behavior of dissociated cells combined with it in culture. Perhaps physical methods of isolation [7] satisfactory for culture purposes could be developed. Such in vitro juxtaposition of cells and matrix material should lead to a fuller description of specific cellular responses and hence a better understanding of tissue organization and stability. This work was supported by grants from the NSF (BMS 73-07022 AOl). and The Universitv of Chicago Cancer Research Center (CA-14599). It-was carried out with the technical assistance of Lindy Andersen and Virginia Kriho.
REFERENCES 1. Bard, J B L & Hay, E, J cell biol67 (1975) 400. 2. Bemfield, M R, Banejee, S D & Cohn, R H, J cell biol 52 (1972) 674. 3. Carlson, E C & Evans, D K, Anat ret I75 (1973) 284. 4. Cohen, A M &Hay, ED, Dev bio126 (1971) 578. 5. DiPasquale, A, Exp cell res 94 (1975) 191. 6. - Ibid 95 (1975) 425. 7. Edds, M V & Sweeny, P R, Synthesis of molecular and cellular structure (ed D Rudnick) p. 11I. Ronald Press, New York (l%l). 8. Fox, H, Arch biol83 (1972) 373. 9. Harrison, R G, Proc sot exp biol NY 4 (1907) 140. IO. Hay, E D, Am zool 13 (1973) 1085. 11. Kamovsky, M J, J cell biol27 (1%5) 137a. 12. Kelly, A M & Chacko, S, Dev biol48 (1976) 421. 13. Manasek, F J, Current topic dev biol 10 (1975) 35.
Culture on basement lamella 14. Meier, S & Hay, E, Dev bio138 (1974) 249. 1.5. Middleton, C A, Locomotion of tissue cells, Ciba foundation sympsoium 14, p. 251. Associate Scientitic Publishers, Amsterdam (1973). 16. Moscona, A, Exp cell res 3 (1952) 535. 17. Nieuwkooo. P D & Faber. J. Normal table of Xenopus ‘i&is. North-Holland, Amsterdam (1956). 18. bverton, J, J morph01 150 (1976) 805. 19. Overton, J & Collins, J, Dev bio148 (1976) 80. 20. Pearlstein, E, Nature 262 (1976) 497. 21. Reddi, A H & Anderson, W A, J cell bio169 (1976) -_5SI. 22. Slavkin, H C, Bringas, P, Cameron, J, LeBaron, R
23. 24. 25. 26. 27. 28.
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& Bavetta, L A, J embryo1 exp morph01 22 (1%9) 395. Trelstad, R L, J histochem cytochem 21 (1973) 521. Tsai, L-J & Overton, J, Dev bio152 (1976) 61. Usuku, G &Gross, J, Dev biol 11 (1965) 352. Waymouth, C, In vitro 10 (1974) 97. Weiss, P & Garber, B, Proc natl acad sci US 38 (1952) 264. Yamada, K M, Yamada, S S & Pastan, I, Proc natl acad sci US 73 (1976) 1217.
Received September 8, 1976 Accepted November 3, 1976
Exp
Cell
Res 105 (1977)
Experimental Cell Research 105 (1977) 3 13-323
RESPONSE CELLS
OF EPITHELIAL TO CULTURE
OBSERVED
AND
MESENCHYMAL
ON BASEMENT
BY SCANNING
LAMELLA
MICROSCOPY
JANE OVERTON Whitman Laboratory,
University of Chicago, Chicago, IL 60637, USA
SUMMARY The basement lamella of Xenopus tadpole skin has been viewed in situ by scanning microscopy, then isolated by trypsin treatment and used as a substrate for cell culture. The basal lamina may also be viewed after EDTA treatment. Responses of epithelial and mesenchymal cells to the lamella have been compared. Mesenchymal cells from chick skin and heart ventricle flatten and attach between the plies of the lamella, then infiltrate it. Myoblasts appear to move less readily within the lamella. Embryonic Xenopus skin epithelium spreads over the surface. Isolated chick skin epithelial cells first begin to spread, then round up and eventually attach to each other in clusters which form a flat basal surface above the lamella. Thus epithelial and mesenchymal cells cultured on this isolated extracellular material mimic aspects of normal tissue organization.
There is increasing interest in the role of the extracellular matrix in development [ 10, 13, 231. However, there are relatively few studies such as those of Meier & Hay [14], Slavkin et al. [22] or Reddi & Anderson [21] in which cell-free matrix material in its original conformation has been recombined with living cells. In the present work, embryonic epithelial or mesenchymal cells have been cultured on the basement lamella of amphibian larval skin in order to assay differences in response to this many layered orthogonal array of collagen fibers. The behavior of cells as indicated by cell shape, alignment or distribution has been followed in an attempt to distinguish differences that may not be apparent under more conventional tissue culture conditions. Collagen fiber systems have been isolated from embryonic chick skin [19], chick duodenum [24] and larval amphibian skin
[18] by a heavy trypsin treatment which removes cellular tissue components, after which the fiber conformation may be viewed by scanning microscopy. This procedure appears to leave unaltered the main course of fiber direction. When the technique is applied to the tadpole the entire basement lamella is exposed [18]. Collagen fibers in these cell-free preparations maintain their orthogonal arrangement. The only apparent divergence from the spatial arrangement found in intact tissue is a variable degree of lateral adhesion between adjacent parallel fibers. The flat lateral surface of the tadpole tail serves as a convenient substrate for cell culture. MATERIALS
AND METHODS
Tadpoles of Xenopus laevis were raised in the laboratory [17] to stages 4%52 when their tails were excised and used as a source of basement lamella. For isolation Exp
Cell
Res
105 (1977)
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J. Overton
Fig. 1. Intact Xenopus tail which has been dissected after drying. E, epidermis; L, basement lamella; I, basal lamina. x 3 400.
Fig.
of the lamella bv EDTA treatment tails were rinsed briefly in penicillin-streptomycin mixture (Microbiological Associates, 5000 U/ml), then incubated 16 h in calcium-magnesium free Hanks’ solution (CMF) containing 0.5 % EDTA. The epithelium could then be stripped off either with forceps or by a 3 min treatment in an ultrasonic cleaning apparatus (L-2R 320 Ultrasonic). Cell-free preparations- were obtained in some cases by this method, but results were not sufficiently reliable to be used for extensive experiments, so this approach was abandoned. For treatment with trypsin, tails were fixed 30 min or more in 70% alcohol, brought gradually to water, then incubated 30 min in 0.1% trypsin (I : 250, GIBCO) in Hanks’ solution adjusted to pH 7.8 and finally rinsed in water. This procedure routinely produced cell-free lamellae. After lamellae were exposed, tails were placed in culture medium on a glass slide in a 35 mm Petri dish. They were positioned with the right side upwards and held in place with glass bridges. For culture of amphibian tissues, the axial region of stage 26 embryos was excised and explanted onto the tail in culture medium consisting of Niu-Twitty solution. For culture of chick skin, tissue was removed from the back of 7 dav embrvos. incubated 6 min at 4°C in 2 % trypsin (1 : 250) in Hanks’ solution after which the eoithelium could be lifted off. Separated eithelial and mesenchymal components were dissociated by incuba-
tion in 0.1% trypsin and 0.5 % EDTA (1 : 250) in CMF for 30 min at 37°C. After rinsing 3 times in Hanks’ solution. cells were disoersed in culture medium bv gentle pipetting. The dispersed cells were then pipette8 onto the positioned tail. For culture of 8 or 9 day chick heart ver&cle cells, excised tissue was dissociated in the same wav as skin. Culture medium for chick cells consisted ofieibovitz L-15 medium (GIBCO) with 0.5 mgl ml L-elutamine. 10% fetal calf serum (GIBCO), 50 Ul ml each of penicillin and streptomycin’, and 0. i mg/ml deoxvribonuclease (Worthinnton). After culture, preparations were fixed in Kamovskv’s fixative fill. followed bv nost-fixation in 1% osmium tetroxidd’ in collidineV buffer. Preparations were then dehydrated in a series of alcohols and amyl acetate, dried in a Tousimis critical point apparatus using CO, as the transitional fluid and coated with gold-palladium in a Hummer gold vapor coater. Preparations were viewed with an ETEC Autoscan. For observation of the basement lamella and basal lamina in situ, intact tails were fixed, dehydrated and dried as described above, then dissected with a fine tungsten needle before coating. For transmission microscopy, tissue fixed in Karnovsky’s fixative and osmium, as described above, was dehydrated in a series of alcohols and propylene oxide, embedded in Araldite, sectioned with adiamond knife on a Porter-Blum MT2 ultramicrotome and viewed with a Hitachi HU-l1A electron microscope.
Exp Cell
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2. Higher magnification of area indicated by the arrow in fig. I. X20000.
Culture
Fig. 3. Denuded surface of Xenopus tail after EDTA treatment. X20000. Fig. 4. Epithelial outgrowth fromXenopus embryo explant onto tail denuded with EDTA. x6000.
RESULTS The substrate
The basement lamella viewed in situ (figs 1 and 2) shovirs a very regular spacing of orthogonally disposed layers of collagen fibers. Some fibers have been disarranged as a result of dissection. The collagen fibers appear to adhere tightly to the basal lamina (fig. 2). The basal lamina has a textured appearance, but no regular substructure has been resolved.
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5. Denuded surface of Xenopus tail after trypsin treatment. X20000. Fig. 6. Epithelial outgrowth fromXenopus embryo explant onto tail denuded with trypsin. x 10000. Fig.
Methods for separating epithelial cells from tissue have shown that with use of EDTA the basal lamina is left behind [lo] whereas it is removed with trypsin [2,4]. In the present experiments, EDTA treatment left a rough surface (figs 3 and 4) with occasional gaps through which the underlying collagen fibers of the lamella could be observed (fig. 4). Viewed at a higher magnification (fig. 3), the lamina shows many small pits, and is obviously imperfectly Exp Cdl
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preserved by the methods used here. Yet a comparison of this surface with the lamina as seen in situ (fig. 2) indicates that a substantial tissue component remains. After trypsin treatment the orthogonal array of collagen fibers can be clearly seen and four or more plies can be distinguished when viewing from the external surface (fig. 5). Individual collagen fibers tend to adhere laterally forming groups of as many as 8 or 10 in some cases. This clustering occurs after trypsin treatment and seems to be present to about the same degree after critical point drying as judged by inspection of sectioned material with transmission microscopy [18]. As a result, the spaces between fibers to which a cultured cell on this surface is exposed may be roughly 200 mm. Observation by transmission microscopy of sectioned trypsinized tails indicates that cells have been removed from the inner region of the tail as well as from the surface. Pigment granules of the chromatophores remain and serve as a convenient marker of the proximal surface of the lamella. Xenopus embryonic epithelium Embryonic axial regions grown as explants on basal lamina or basement lamella as prepared here send out epithelial sheets which extend over the surface of the substrate. At the free edge of such outgrowths on the lamina, flattened lamellipodia bear processes which show no particular relation to the collagen arrangement which lies beneath (fig. 4). On the lamella, cell processes tend to follow the direction of the superficial collagen fibers (fig. 6). No ruffling was seen. In both instances considerable areas of the surface may be covered with cells. Because of the difficulty encountered in preparing clean surfaces of larnina, other cell types were grown only on the lamella. Exp Cell
Res IOS (1977)
Chick skin Epithelial and mesenchymal skin components were detached and cultured separately as dispersed cells on the lamella. By 2 h mesenchymal cells had flattened on the surface and typically had sent out a number of broad cell processes which were buried beneath the collagen at their terminal regions (fig. 7). By 4 h (fig. 8) cells were spread extensively and often buried deeper. Cellular extensions tended to be closely aligned with lower plies of collagen. A cross-section of the lamella viewed by transmission microscopy at 16 h shows mesenchyme cells layered between the plies (fig. 9). Although the collagen maintains its orthogonal arrangement over this period in some regions, in the vicinity of cells it tends to become disorganized (cf figs 8 and 10). Viewing such a preparation from the surface, it is evident that many cells have moved into the interior of the lamella. Cells not only move into but through the lamella (fig. 9). They are commonly found by 16 h in the interior of the fin. In all the stages of cell spreading and dispersion which were examined, cell bodies and cell processes were characteristically flattened with no evidence of blebbing or ruffling. Many epidermal cells have begun to spread after 2 h of culture on the lamella and their processes tend to align with the collagen fibers (fig. 11). These cells can be compared with cells in the same culture which have settled on the coverslip rather than on the surface of the fin. The cells on glass are somewhat more flattened. By 4 h, cells on glass have continued to flatten, while those on the lamella, in contrast, have become more rounded (fig. 12). Cell processes are now largely or completely withdrawn, but points of attachment to the collagen fibers persist and fiber organization in
Culture on basement lamella
Fig. 7. Chick skin mesenchyme cell on basement lamella, 2 h. x4000. Fig. 8. Mesenchyme cell on basement lamella, 4 h. x2ooo. Fig. 9. Thin section showing mesenchyme cells within pigand beneath the basement lamella. I6 h. Arrow, 21-771815
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ment granules from a Xenopus chromatophore which mark the inner surface of the basement lamella. X15WO. Fig. IO. Mesenchyme cells within the basement lamella, 6 h. x2000.
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the immediate vicinity of the cells is disturbed. These compact cells characteristically have one or more large rounded blebs. By 16 h (figs 13 and 14) cells are usually found in clusters at the surface. Although the orthogonality of the lamella is maintained at a distance from these clusters, in regions near the cells, the pattern is distorted. In cross-sections of the lamella viewed by transmission microscopy (fig. 13) the basal surface of the clusters is seen as smooth and flattened, resting above the collagen. Nuclei are elongated and lie close together, and the cells of this reconstructed epithelial fragment have relatively little surface exposed to the collagen. Beneath these clusters, the collagen is always distorted and frequently lies in accordion-like pleats. A suggestion of such an arrangement is seen in fig. 13. No evidence of a basal lamina was seen beneath these epithelial clusters, but the longest culture period was 16 h, and observations of others [3, 41 indicate that l-2 days are usually required for reformation of the lamina. Chick heart ventricle The heart ventricle contains cells of a number of types but myoblasts and fibroblasts on which attention is focused here are in the majority. After 2 h of culture on the lamella, suspensions of heart cells had given rise to cells of two characteristic conformations (fig. 15) interpreted here as myoblasts and libroblasts. Fibroblasts had begun to flatten and spread by sending out a number of broad processes which had been inserted beneath the upper ply of collagen and tended to be aligned with lower plies. Their conformation and behavior was similar to skin libroblasts throughout the period studied, They could be seen at 16 h within the lamella, often aligned between plies, and by this time in numerous instances they Exp CeNRes
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were found beneath the lamella as well. Myoblasts which could be identified with certainty by transmission microscopy when contractile elements were seen, showed a very different behavior. They tended to adhere near the surface of the lamella as judged by a transmission microscopy study of sectioned material. Bipolar cells which were rounded in cross-section, seen at 2 h (figs 15 and 16), could also be seen near the surface at 16 h (figs 17 and 18). At 2 h these bipolar cells showed a very strict alignment with the substrate. Orientation is clearly parallel to the second ply and at right angles to the first. Frequently the majority of the contractile elements in the cytoplasm of a myoblast ran parallel to nearby collagen bundles (fig. 17). The orthogonality of the lamella was distorted by 16 h, perhaps due to contractions of myoblasts as well as activity of fibroblasts (cf figs 16 and 18). Thus, though these cells may send processes deep into the collagen and become covered by collagen fibers, they do not appear to move as freely within and beneath the lamella as do fibroblasts.
DISCUSSION Early attempts to disaggregate cells employing trypsin [26] led to the development of a technique for dissociating and reassociating cells from tissues of higher vertebrates [ 161 which has become a widely used method for analysis of tissue organization. In the present study, cellular and non-cellular tissue components are trypsin dissociated and recombined. Just as reassociated cells give rise to normal tissue patterns, these experiments with cellular and mimic non-cellular tissue components aspects of normal tissue organization; epithelial cells form a smooth basal surface
Culture on basement lamella
II. Chick skin epithelial cell on basement lamella, 2 h. x5000. Fig. 12. Skin epithelial cells on basement lamella, 4 h.
Fig.
x2ooo.
3 19
13. Thin section of skin epithelial cells resting on top of basement lamella. ~4 500. Fig. 14. Cluster of skin epithelial cells on outer surface of basement lamella. x I 500.
Fig.
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Fig. IS. Cells from chick heart ventricle on basement lamella, 2 h. E, presumed fibroblast. M, presumed myoblast. x2 300. Fig. 16. Bipolar ventricle cell showing strict alignment with fibers of basement lamella, 2 h. X2 500.
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Fig. 17. surface Fig. 18. at 16 h.
Myoblasts seen in thin section lying near the of the basement lamella, 16 h. X 14000. Presumed myoblasts near the lamella surface x4000.
Culture
above the collagen and mesenchymal cells invade and penetrate it. Epithelial cells of the Xenopus embryo grow across the surface of the basement lamella in a sheet the leading edge of which sends out processes which show a tendency to align with collagen fibers of the lamella. Likewise, trypsin dissociated epithelial cells of the chick skin, early in culture, show a tendency to flatten on the surface and send out microspikes which frequently follow the direction of adjacent fibers. These initially flattened cells then round up, and by 4 h in culture show a number of large hemispherical blebs. In contrast, cells in these same cultures which have settled on the glass coverslip continue to flatten. By 16 h cells on the lamella which have made contact with each other are associated in clusters. Scanning images suggest that the initially flattened cells make contact with the collagen fibers and move about, distorting the orthogonal pattern. As contact between cells in clusters increases, reducing the cell surface facing the collagen, the lamella appears to become compressed forming irregular pleats. It is possible that the distortion of the normal lamellar pattern is caused not only by physical forces exerted by the cells but also by digestion, but there is no clear evidence for this. The behavior of these epithelial cells is reminiscent of reports in the literature that isolated epithelial cells, as opposed to epithelial cells in sheets, have a tendency to round up. Observation of living cultures of chick skin, intestinal and cornea1 epithelial sheets grown on glass indicate that cells which leave a spreading sheet round up and bleb [5, 61. Also, chick pigmented retina epithelium cells dissociated and plated out on reconstituted collagen are poorly spread and show much blebbing early in the life of the culture [15]. This tendency to round up
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presumably is one factor which contributes to keeping skin epithelial cells on the surface of the lamella. Why the clustered cells in the present experiments fail to spread is not clear. Possibly the weakened and distorted organization of the collagen in the vicinity of the clusters will not support spreading. At least one factor which controls the distribution of these epithelial cells on the lamella appears to be the strong tendency for the cells to adhere to each other [5]. Mesenchymal cells in all cases, skin fibroblasts, heart fibroblasts and heart myoblasts, always showed a tendency to penetrate the lamella. These cells all have flattened extensions and in the case of fibroblasts, the cell bodies are also extremely flattened. Cell processes show a pronounced orientation in accordance with the organization of the substrate. Cell processes, and eventually whole cells, move between the plies of the basement lamella and fibroblasts may even move through it to the proximal side. In the living amphibian, mesenchymal cells of the skin also penetrate the basement lamella of the tail at metamorphosis. Fine structure studies of the process in normal metamorphosis [8] and in hormone-treated tadpoles [25] show that the invading cells may be layered between the plies as in the case described here. At metamorphosis the basement lamella becomes increasingly loosened and frayed, and the invading cells phagocytize large amounts of collagen, There was no evidence in the present study that collagen was phagocytized. It is possible, as in the case of epithelial cells, that it is broken down enzymatically. In any event, its organization is considerably distorted by the end of 16 h of culture in the presence of these cells. With each cell type studied here there Exp Cdl
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was evidence that cell processes were guided by the orientation of the substrate. This aspect of cell behavior has been recognized for a long time [9]. However, the various cell types showed great variability in the degree of response they exhibited. The most striking orientation was seen in myoblasts where a bipolar cell body was oriented parallel to the second ply and at right angles to the first. Cardiac myoblasts grown on a collagen gel are not necessarily bipolar [12], but these cells do exhibit the greatest polarity of those used in this study. Highly polarized cells showed the most orientation and possibly, the conformation of the basement lamella contributed to the extreme degree of polarization [27]. Polarization of a cell or its capacity to become polarized may be correlated with its capacity to infiltrate the lamella. But this is not the only important factor, since fibroblasts which tended to be less polarized infiltrated the lamella more extensively than myoblasts. Ruffling of the cell surface which is a common type of surface movement of cells in culture, was almost completely absent under the culture conditions used here. This is consistent with the observations of Bard & Hay [I] that cells moving through their normal collagen matrix do not exhibit this surface behavior. DiPasquale [5] has compared the behavior of embryonic epithelial and mesenchymal cells and points out that in both cases lamellipods, ruffles and blebs are formed, although to different extents. The kinds of behavioral differences studied in the present work will presumably be clarified in part by further studies of plasma membrane glycoprotein which mediates adhesion of fibroblasts to collagen [20, 281. The culture of cells on collagen components isolated from tissue as in this study not only shows a very different response in E.rp Cd
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epithelial and mesenchymal cells, but elicits behavior different from that seen when cells are grown on artificial substrates. For example, epithelial cells grown on filters will typically send fine cell processes deep into the underlying cavities. The method of isolating the basement lamella with trypsin which was used here obviously left a structure depleted of some components. In addition, the lamella was distorted since collagen fibers which are normally evenly spaced were sometimes clumped to give intervening spaces of -200 nm. Furthermore, the basal lamina had been removed. It is possible that isolation of this intercellular region in a more intact state would evoke a different behavior of dissociated cells combined with it in culture. Perhaps physical methods of isolation [7] satisfactory for culture purposes could be developed. Such in vitro juxtaposition of cells and matrix material should lead to a fuller description of specific cellular responses and hence a better understanding of tissue organization and stability. This work was supported by grants from the NSF (BMS 73-07022 AOl). and The Universitv of Chicago Cancer Research Center (CA-14599). It-was carried out with the technical assistance of Lindy Andersen and Virginia Kriho.
REFERENCES 1. Bard, J B L & Hay, E, J cell biol67 (1975) 400. 2. Bemfield, M R, Banejee, S D & Cohn, R H, J cell biol 52 (1972) 674. 3. Carlson, E C & Evans, D K, Anat ret I75 (1973) 284. 4. Cohen, A M &Hay, ED, Dev bio126 (1971) 578. 5. DiPasquale, A, Exp cell res 94 (1975) 191. 6. - Ibid 95 (1975) 425. 7. Edds, M V & Sweeny, P R, Synthesis of molecular and cellular structure (ed D Rudnick) p. 11I. Ronald Press, New York (l%l). 8. Fox, H, Arch biol83 (1972) 373. 9. Harrison, R G, Proc sot exp biol NY 4 (1907) 140. IO. Hay, E D, Am zool 13 (1973) 1085. 11. Kamovsky, M J, J cell biol27 (1%5) 137a. 12. Kelly, A M & Chacko, S, Dev biol48 (1976) 421. 13. Manasek, F J, Current topic dev biol 10 (1975) 35.
Culture on basement lamella 14. Meier, S & Hay, E, Dev bio138 (1974) 249. 1.5. Middleton, C A, Locomotion of tissue cells, Ciba foundation sympsoium 14, p. 251. Associate Scientitic Publishers, Amsterdam (1973). 16. Moscona, A, Exp cell res 3 (1952) 535. 17. Nieuwkooo. P D & Faber. J. Normal table of Xenopus ‘i&is. North-Holland, Amsterdam (1956). 18. bverton, J, J morph01 150 (1976) 805. 19. Overton, J & Collins, J, Dev bio148 (1976) 80. 20. Pearlstein, E, Nature 262 (1976) 497. 21. Reddi, A H & Anderson, W A, J cell bio169 (1976) -_5SI. 22. Slavkin, H C, Bringas, P, Cameron, J, LeBaron, R
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& Bavetta, L A, J embryo1 exp morph01 22 (1%9) 395. Trelstad, R L, J histochem cytochem 21 (1973) 521. Tsai, L-J & Overton, J, Dev bio152 (1976) 61. Usuku, G &Gross, J, Dev biol 11 (1965) 352. Waymouth, C, In vitro 10 (1974) 97. Weiss, P & Garber, B, Proc natl acad sci US 38 (1952) 264. Yamada, K M, Yamada, S S & Pastan, I, Proc natl acad sci US 73 (1976) 1217.
Received September 8, 1976 Accepted November 3, 1976
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