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Effect of vitamin A on epithelial morphogenesis in vitro
Copyright 0 1973 by Academic Press, Inc. AN rights of reproduction in any form reserved
Experimental Cell Research 76 (1973) 118-126
EFFECT
OF VITAMIN
A ON EPITHELIAL
MORPHOGENESIS
IN VITRO Fine Structural
Changes in Explants M. L. BARNETT
Department
of Periodontology
of Adult Mammalian
Skin
and G. SZABO
and Oral Histopathology, Harvard Boston, Mass. 02115, USA
School
of Dental Medicine,
SUMMARY
Explants of adult mammalian skin were maintained in culture with either high levels of vitamin A or control media. Control explants retained characteristics of a keratinizing epithelium. Treated explants underwent marked changeswhich included the formation of microvilli with a filamentous surface coating, inhibition of keratinization, decreasein number and size of tonofilament bundles and desmosomes,widening of intercellular spaces,and appearanceof tightjunction-like complexes.
Vitamin A is of interest to developmental biologists because it is a chemical having a specific morphogenetic effect on epithelium. It was originally observed [l] that in 7-day chick embryo skin explants cultured in a medium containing high levels of vitamin A, keratinization is completely suppressed within 7 to 10 days and, moreover, the epithelium is transformed into a mucous-secreting type. This transformed epithelium is virtually identical histologically to chick nasal epithelium of comparable age [I]. However, the effect of the vitamin is reversible so that keratinization reappears following transfer of the explants to normal medium [l]. Subsequent studies, also on embryonic chick skin, have further characterized the nature of the tissue response. These have demonstrated an inverse relationship between the age of the embryonic tissue and the degree of response [2], and have, with autoradioExptl
Cell Res 76 (1973)
graphic techniques, identified a decrease in keratin production and more widespread utilization of sulphate throughout the treated tissue when compared to controls [3]. The fine structure of the treated epithelium has been described during both mucus metaplasia and its reversal [4]. The transformed epithelium is characterized by a loss of tonofilaments and inhibition of keratinization, a disorientation of basal and lower stratum spinosum cells with an increase in size of intercellular lacunae, a general lack of cell density, and the formation of microvilli at the outer surface of the epidermis [4]. Mucus globules were also seen in some of the treated cells. Of interest was the finding of both tonofilaments and mucus globules within the same cell in the middle strata of treated epithelium which had been returned to normal medium [4]. More recently, it was shown [5] that an in vitro effect of vitamin A can
In vitro effect of vitamin A on epithelium be obtained using a synthetic medium (BGJ) instead of the plasma clot-embryo extract medium which was originally used. Investigations into the effect of vitamin A on mammalian skin have produced less dramatic, and rather variable, results. These studies have most often employed local administration of large doses of the vitamin to adult skin in vivo and have produced findings ranging from a minimal effect [6] to epithelial hyperplasia, alone or in combination with other abnormalities [7-91. In vitro studies on embryonic mammalian skin [lo, II] have shown that vitamin A may affect epithelial morphogenesis, but that its action is both species and site dependent. No definitive information regarding the in vitro effect of hypervitaminosis A on adult ‘committed’ mammalian epidermis has heretofore been presented, although evidence suggestive of an action has been reported [12]. The purpose of this study was to investigate the morphogenetic effect of high levels of vitamin A on adult mammalian epidermis in vitro, with particular reference to fine structural changes.
MATERIALS
AND METHODS
Explants of skin, consisting of the superficial dermis and whole epidermis, wereobtained from the dorsum of the ear of adult guinea pigs and cultured, with the epidermis upwards;using a modified raft technique. The explant was placed on a square of Millipore filter, which in turn rested on a segment of millioore orefilter pad immersed in either gaggleminimum essential medium (BBL Division of Bioquest, Cockeysville, Md) with 10 % calf serumorBGJmedium[l3](GIBCo, Grand Island, N.Y.) with 10% horse serum added. The experimental cultures contained, as well, 22-30 IU vitamin A alcohol (Sigma Chemical Co., St Louis, MO.) dissolved in ethanol, per ml of medium, whereas the control cultures contained an equivalent amount of the ethanol diluent alone. The cultures were gassed with a mixture of 5 % CO, in air, and incubated at 37°C. The medium, with appropriate modifications. was changed every 2 to 3 days. The vitamin A stock solution was refrigerated under nitrogen to minimize oxidation of the vitamin. At the time of explantation, and after 3,6 and 10 days in culture, explants were fixed in Ito-Karnovsky
119
fixative [14] for 2 h, post-fixed in osmium tetroxide for 1 h, dehydrated in graded ethanols, and embedded in Epon [15]. Thick sections for surveying were stained with toluidine blue. Thin sections were stained with uranyl acetate and lead citrate [16], and studied with an RCA EMU-3G electron microscope.
RESULTS Controls The epidermis at time of explantation was a keratinizing stratified squamous epithclium, approx. 8 cells in thickness (fig. 1). A rather prominent stratum granulosum was seen. The dermal-epidermal junction was smoothly undulating, with very short rete ridges present. Several types of epithelial cells were present, including keratinocytes and cells comprising the pilosebaceous unit. No sweat glands were seen. The epidermis also contained dendritic cells. These included melanocytes in the basal level, and higher level clear dendritic cells, many of which could be identified ultrastructurally as Langerhans cells by virtue of their characteristic granules. On a fine structural level, features characteristic of a keratinizing epithelium were present, including tonofilament bundles, desmosomes, membrane coating granules, keratohyalin granules, and membrane changes associated with the stratum corneum. Cells in all layers were in close apposition to one another. In the basal layer, interdigitating cell processes held together by desmosomes could be identified. Intercellular spaces although identifiable were relatively narrow. At higher levels, intercellular spaces progressively narrowed and longer and more frequent desmosomes were seen. Melanosomes were present in both melanocytes and keratinocytes. The control explants retained many of the features of the original epithelium, even after 10 days in culture (figs 2, 3). Tonofilaments, desmosomes, and keratohyalin were still formed, and in some explants a cell layer Exptl Cell Res 76 (1973)
120 M. L. Barnett & G. Szabo appearing similar to stratum corneum could be identified. However, membrane coating granules were not observed. The cells remained in close apposition to each other, although cell borders becamerather straight. Melanosomes were present, either free within the cytoplasm or contained within lysosomal structures. Thus, although some changes had occurred in the control explants as a result of culturing, they still retained features characteristic of a keratinizing epithelium.
Vitamin A-treated explants The explants cultured with high levels of vitamin A were markedly different from the controls. By 6 days in vitro there were no apparent signs of overall keratinization, although small bundles of tonofilaments were occasionally present. Desmosomes also were infrequent and short. The cells did not flatten
as they approached the free tissue surface, but remained rounded. The most striking change was the formation of numerous regularly spaced microvilli on both the free and internal cell surfaces (fig. 4). At higher magnifications, these microvilli were covered by a filamentous coating which was perpendicular to the cell surface and in apparent intimate association with the outer leaflet of the cell membrane (fig. 5a). This coating was morphologically similar to the enteric coat, a specific form of glycocalyx described on intestinal microvilli [17]. On occasion, microvilli were seen which had an internal skeletal substructure (fig. 5b). These changes were not restricted to the free tissue surface, but were also seen deeper within the epithelium. Intercellular spaces were widened, and structures reminiscent of canaliculi were formed (figs 6, 7). At the periphery of many of these widened spaces,
Fig. 1. Adult guinea pig skin at time of explantation. A survey electron micrograph of the keratinizing epithelium showing prominent bundles of tonofilaments, keratohyalin granules (kh), and stratum corneum (SC).The cells flatten as they approach the surface. All cells are in close apposition to each other. x 1 700. Fig. 2. Six-day control explant. Tonofilaments and desmosomes can be seen, as well as a layer resembling stratum corneum (SC).A degenerating ‘ballooned’ cell is uresent at the surface. Cells are flattened. with straight borders, and minimal intercellular spaces are visible. Numerous melanosomes (mel) are present.’ x 3 000. Fig. 3. Ten-day control explant. Keratohyalin (MI) is present in addition to tonofilaments and desmosomes. The cell membrane at the-free surface of the epithelium is smooth and free of an attached surface coating. x9600. Fig. 4. Six-day vitamin A-treated explant. Regularly spaced microvilli are present on the free cell surface. In some regions, an associated filamentous surface coating can be discerned. Tonofilaments are present in small bundles, and desmosomes are rare. There appears to be a widening of the intercellular space. A junctional complex suggestive of a tight junction (t) is also present. A Golgi zone (g) can be identified. x 39 000. Fig. 5. Ten-day vitamin A-treated explant. Well developed microvilli with a prominent filamenrous surface coat in intimate association with (a) the outer leaflet of the cell membrane, and (b) with a skeletal substructure are identified. x 70 000. Fig. 6. Ten-day vitamin A-treated explant. Intercellular spaces have widened and microvilli have formed several cell layers deep to the epithelial surface. Desmosomes are rare, and tonofilaments although present are not conspicuous. Numerous melanosomes can be seen, both free within the cytoplasm and contained with lysosomal structures. x 8 000. Fig. 7. Six-day vitamin A-treated explant. A higher magnification of a widened intercellular space showing microvilli with an associated filamentous surface coat. Tonofilaments are present in small bundles but are rarely seen close to the cell periphery. Desmosomes appear to be breaking down. The overall structure is reminiscent of a canaliculus. x 39OOG. Fig. 8. Six-day vitamin A-treated explant. An intercellular junction resembling a tight junction (0) has appeared at the periphery of a widened intercellular space. Projecting microvilli with a filamentous coating are prominent. x70000. Fig. 9. Six-day vitamin A-treated explant. Cells b and c appear to be undergoing a morphogenetic change in response to vitamin A, whereas cell a is not responding. Remnants of desmosomal plaques (dp) which presumably were formerly apposed can be identified. Incell c there is a progressive increase in the extent of microvillus development. A filamentous surface coat is present on the microvilli of cells b and c but is not present on the surface of cell a. The remaining desmosomes appear to be breaking down. x 39 000. Exptl Cell Res 76 (1973)
In vitro effect of vitamin A on epithelium 121
Exptl Cell Res 76 (1973)
122 M. L. Barnett & G. Szabo
Exptl Cell Res 76 (1973)
In vitro effect of vitamin A on epithelium
123
Exptl Cell Res 76 (1973)
124 M. L. Barnett & G. Szabo
Exptl Cell Res 76 (1973)
In vitro effect of vitamin A on epithelium junctional complexes resembling tight junctions (zonula occludens) [18] could be identified, thus enhancing the resemblance to a glandular structure (fig. 8). Other changes which were seen included an apparent increase in extent of Golgi regions, and small vesicles, found both within the cytoplasm and in association with the cell membrane. No membrane coating granules were present. One section (fig. 9) was particularly interesting because it suggested a sequence of events which may result in the characteristic surface changes. Three epithelial cells are shown, two of which appear to be responding to vitamin A, whereas the uppermost one appears not to have responded. Remnants of formerly apposing desmosomal plaques can be seen in all cells. However, in the responding cells microvilli, with a filamentous surface coat, show progressively better formation as they get farther away from the desmosomal remains. This suggests that an initial step in the morphogenetic change is a destruction of desmosomes and, in responding cells, attachment plaques as well, with subsequent retraction and loss of tonofilaments and microvillus formation. As desmosomes are lost, intercellular spaces widen. Other changes, including the appearance of ‘tight junctions may be simultaneous with, or subsequent to, these initial cellular alterations. DISCUSSION The morphologic changes described above and the absence of any signs of keratinization in the treated explants indicate that vitamin A can, indeed, influence the morphogenesis of adult mammalian epidermis in vitro. Our findings are consistent in many respects with those of a previous ultrastructural study on altered embryonic chick skin [4]. In particular, the findings of a decrease
125
in the size of tonofilament bundles, the formation of intercellular lacunae and microvilli, and a general decrease in cell density have been mentioned as being characteristic of transformed chick skin [4]. In addition, mucus globules were described in treated chick epidermis [4]. Whereas such globules were not seen in our sections, the formation of microvilli containing a well developed surface coating and the appearance of tightjunction-like complexes indicates that the tissue has undergone a significant transformation. Of perhaps greater interest is a comparison of the findings described herein with those from a study on in vivo squamous metaplasia of adult tracheal epithelium in vitamin Adeficient rats [19]. Our epithelial cells, transforming from a keratinizing type to a mucous type, share a great many features with tracheal cells which are midway along the path of squamous metaplasia from a respiratory type. These results, in combination with past findings, suggest that adult mammalian epithelia may show a continuum of morphogenetic responses to vitamin A, depending upon the threshold of a given epithelium
[l, 201. Whereas vitamin A per se may not be involved in an inductive interaction between adult dermis and epidermis in vivo, the fact that adult epithelium exposed to an exogenous agent can undergo the marked changes described above also lends indirect support to the theory for the conservation of epithelia1 specificity which states that the pathway of differentiation in any given region followed by presumably equipotential germinative epithelial cells is dependent upon a specific inductive stimulus from the underlying dermis
1211. Although early changes in vitamin Atreated cells appear to involve desmosomal Exptl Cell Res 76 (1973)
126 M. L. Barnett and G. Szabo complexes, the exact mechanism of action of the vitamin in producing this change is unknown. In view of the widespread changes seen in the treated tissue, it does not seem unreasonable to assume that the agent in some way ultimately affects gene expression [22,23]. Certainly, several possibilities exist by which this can occur, either directly or indirectly. However, the mechanism by which epithelial morphogenesis is affected remains to be elucidated. This study was supported by USPHS Grants DE00111 and DE-01766 from the National Institute of Dental Research.
REFERENCES 1. Fell, H B & Mellanby, E, J physiol 119 (1953) 470. 2. Fell, H B, Proc roy sot London, ser B 146 (1957) 242. 3. Pelt, S R & Fell, H B, Exptl cell res 19 (1960) 99.
4. Fitton Jackson, S & Fell, H B, Dev biol 7 (1963)
7. Bern, H A, Elias, J J, Pickett, P B, Powers, T R & Harkness, M N, Am j anat 96 (1955) 419. 8. Lawrence, D L & Bern, H A, J invest derm 31 (1958) 313. 9. Sabella, J D, Bern, H A & Kahn, R H, Proc sot exptl biol med 76 (1951) 499. 10. New, D A T, Brit j derm 75 (1963) 320. 11. - Exptl cell res 39 (1965) 178. 12. Szabo, G, Fundamentals of keratinization (ed E 0 Butcher & R Sognnaes) D. 45. Amer assoc advancement sci, Waihington-(1962). 13. Biggers, J D, Gwatkin, R B L & Heyner, S, Exptl cell res 41 (1961) 24. 14. Ito, S & Karnovsky, M, J cell biol 39 (1968) 168a. 15. Luft, J H, J biophys biochem cytol 9 (1961) 409. 16. Reynolds, E S, J cell biol 17 (1963) 208. 17. Ito, S, J cell biol 27 (1965) 475. 18. Farquhar, M G & Palade, G E, J cell biol 17 (1963) 375. 19. Wong, Y-C & Buck, R C, Lab invest 24 (1971) 55.
20. Wilde, C E, Cell, organism and milieu (ed D Rudnick) n. 3. The Ronald Press, New York (1959). ’ 21. Billingham, R E & Silvers, W K, J exptl med 125 (1967) 429. 22. deluca, L &Wolf,
G, Am j clin nutr 22 (1969) 1050. 23. Prutkin, L, J invest derm 57 (1971) 323.
394.
5. Rothberg, S, J invest derm 49 (1967) 35. 6. Montagna, W, Proc sot exptl biol med 86 (1954) 668.
Exptl Cell Res 76 (1973)
Received May 23, 1972 Revised version received July 21, 1972
Experimental Cell Research 76 (1973) 118-126
EFFECT
OF VITAMIN
A ON EPITHELIAL
MORPHOGENESIS
IN VITRO Fine Structural
Changes in Explants M. L. BARNETT
Department
of Periodontology
of Adult Mammalian
Skin
and G. SZABO
and Oral Histopathology, Harvard Boston, Mass. 02115, USA
School
of Dental Medicine,
SUMMARY
Explants of adult mammalian skin were maintained in culture with either high levels of vitamin A or control media. Control explants retained characteristics of a keratinizing epithelium. Treated explants underwent marked changeswhich included the formation of microvilli with a filamentous surface coating, inhibition of keratinization, decreasein number and size of tonofilament bundles and desmosomes,widening of intercellular spaces,and appearanceof tightjunction-like complexes.
Vitamin A is of interest to developmental biologists because it is a chemical having a specific morphogenetic effect on epithelium. It was originally observed [l] that in 7-day chick embryo skin explants cultured in a medium containing high levels of vitamin A, keratinization is completely suppressed within 7 to 10 days and, moreover, the epithelium is transformed into a mucous-secreting type. This transformed epithelium is virtually identical histologically to chick nasal epithelium of comparable age [I]. However, the effect of the vitamin is reversible so that keratinization reappears following transfer of the explants to normal medium [l]. Subsequent studies, also on embryonic chick skin, have further characterized the nature of the tissue response. These have demonstrated an inverse relationship between the age of the embryonic tissue and the degree of response [2], and have, with autoradioExptl
Cell Res 76 (1973)
graphic techniques, identified a decrease in keratin production and more widespread utilization of sulphate throughout the treated tissue when compared to controls [3]. The fine structure of the treated epithelium has been described during both mucus metaplasia and its reversal [4]. The transformed epithelium is characterized by a loss of tonofilaments and inhibition of keratinization, a disorientation of basal and lower stratum spinosum cells with an increase in size of intercellular lacunae, a general lack of cell density, and the formation of microvilli at the outer surface of the epidermis [4]. Mucus globules were also seen in some of the treated cells. Of interest was the finding of both tonofilaments and mucus globules within the same cell in the middle strata of treated epithelium which had been returned to normal medium [4]. More recently, it was shown [5] that an in vitro effect of vitamin A can
In vitro effect of vitamin A on epithelium be obtained using a synthetic medium (BGJ) instead of the plasma clot-embryo extract medium which was originally used. Investigations into the effect of vitamin A on mammalian skin have produced less dramatic, and rather variable, results. These studies have most often employed local administration of large doses of the vitamin to adult skin in vivo and have produced findings ranging from a minimal effect [6] to epithelial hyperplasia, alone or in combination with other abnormalities [7-91. In vitro studies on embryonic mammalian skin [lo, II] have shown that vitamin A may affect epithelial morphogenesis, but that its action is both species and site dependent. No definitive information regarding the in vitro effect of hypervitaminosis A on adult ‘committed’ mammalian epidermis has heretofore been presented, although evidence suggestive of an action has been reported [12]. The purpose of this study was to investigate the morphogenetic effect of high levels of vitamin A on adult mammalian epidermis in vitro, with particular reference to fine structural changes.
MATERIALS
AND METHODS
Explants of skin, consisting of the superficial dermis and whole epidermis, wereobtained from the dorsum of the ear of adult guinea pigs and cultured, with the epidermis upwards;using a modified raft technique. The explant was placed on a square of Millipore filter, which in turn rested on a segment of millioore orefilter pad immersed in either gaggleminimum essential medium (BBL Division of Bioquest, Cockeysville, Md) with 10 % calf serumorBGJmedium[l3](GIBCo, Grand Island, N.Y.) with 10% horse serum added. The experimental cultures contained, as well, 22-30 IU vitamin A alcohol (Sigma Chemical Co., St Louis, MO.) dissolved in ethanol, per ml of medium, whereas the control cultures contained an equivalent amount of the ethanol diluent alone. The cultures were gassed with a mixture of 5 % CO, in air, and incubated at 37°C. The medium, with appropriate modifications. was changed every 2 to 3 days. The vitamin A stock solution was refrigerated under nitrogen to minimize oxidation of the vitamin. At the time of explantation, and after 3,6 and 10 days in culture, explants were fixed in Ito-Karnovsky
119
fixative [14] for 2 h, post-fixed in osmium tetroxide for 1 h, dehydrated in graded ethanols, and embedded in Epon [15]. Thick sections for surveying were stained with toluidine blue. Thin sections were stained with uranyl acetate and lead citrate [16], and studied with an RCA EMU-3G electron microscope.
RESULTS Controls The epidermis at time of explantation was a keratinizing stratified squamous epithclium, approx. 8 cells in thickness (fig. 1). A rather prominent stratum granulosum was seen. The dermal-epidermal junction was smoothly undulating, with very short rete ridges present. Several types of epithelial cells were present, including keratinocytes and cells comprising the pilosebaceous unit. No sweat glands were seen. The epidermis also contained dendritic cells. These included melanocytes in the basal level, and higher level clear dendritic cells, many of which could be identified ultrastructurally as Langerhans cells by virtue of their characteristic granules. On a fine structural level, features characteristic of a keratinizing epithelium were present, including tonofilament bundles, desmosomes, membrane coating granules, keratohyalin granules, and membrane changes associated with the stratum corneum. Cells in all layers were in close apposition to one another. In the basal layer, interdigitating cell processes held together by desmosomes could be identified. Intercellular spaces although identifiable were relatively narrow. At higher levels, intercellular spaces progressively narrowed and longer and more frequent desmosomes were seen. Melanosomes were present in both melanocytes and keratinocytes. The control explants retained many of the features of the original epithelium, even after 10 days in culture (figs 2, 3). Tonofilaments, desmosomes, and keratohyalin were still formed, and in some explants a cell layer Exptl Cell Res 76 (1973)
120 M. L. Barnett & G. Szabo appearing similar to stratum corneum could be identified. However, membrane coating granules were not observed. The cells remained in close apposition to each other, although cell borders becamerather straight. Melanosomes were present, either free within the cytoplasm or contained within lysosomal structures. Thus, although some changes had occurred in the control explants as a result of culturing, they still retained features characteristic of a keratinizing epithelium.
Vitamin A-treated explants The explants cultured with high levels of vitamin A were markedly different from the controls. By 6 days in vitro there were no apparent signs of overall keratinization, although small bundles of tonofilaments were occasionally present. Desmosomes also were infrequent and short. The cells did not flatten
as they approached the free tissue surface, but remained rounded. The most striking change was the formation of numerous regularly spaced microvilli on both the free and internal cell surfaces (fig. 4). At higher magnifications, these microvilli were covered by a filamentous coating which was perpendicular to the cell surface and in apparent intimate association with the outer leaflet of the cell membrane (fig. 5a). This coating was morphologically similar to the enteric coat, a specific form of glycocalyx described on intestinal microvilli [17]. On occasion, microvilli were seen which had an internal skeletal substructure (fig. 5b). These changes were not restricted to the free tissue surface, but were also seen deeper within the epithelium. Intercellular spaces were widened, and structures reminiscent of canaliculi were formed (figs 6, 7). At the periphery of many of these widened spaces,
Fig. 1. Adult guinea pig skin at time of explantation. A survey electron micrograph of the keratinizing epithelium showing prominent bundles of tonofilaments, keratohyalin granules (kh), and stratum corneum (SC).The cells flatten as they approach the surface. All cells are in close apposition to each other. x 1 700. Fig. 2. Six-day control explant. Tonofilaments and desmosomes can be seen, as well as a layer resembling stratum corneum (SC).A degenerating ‘ballooned’ cell is uresent at the surface. Cells are flattened. with straight borders, and minimal intercellular spaces are visible. Numerous melanosomes (mel) are present.’ x 3 000. Fig. 3. Ten-day control explant. Keratohyalin (MI) is present in addition to tonofilaments and desmosomes. The cell membrane at the-free surface of the epithelium is smooth and free of an attached surface coating. x9600. Fig. 4. Six-day vitamin A-treated explant. Regularly spaced microvilli are present on the free cell surface. In some regions, an associated filamentous surface coating can be discerned. Tonofilaments are present in small bundles, and desmosomes are rare. There appears to be a widening of the intercellular space. A junctional complex suggestive of a tight junction (t) is also present. A Golgi zone (g) can be identified. x 39 000. Fig. 5. Ten-day vitamin A-treated explant. Well developed microvilli with a prominent filamenrous surface coat in intimate association with (a) the outer leaflet of the cell membrane, and (b) with a skeletal substructure are identified. x 70 000. Fig. 6. Ten-day vitamin A-treated explant. Intercellular spaces have widened and microvilli have formed several cell layers deep to the epithelial surface. Desmosomes are rare, and tonofilaments although present are not conspicuous. Numerous melanosomes can be seen, both free within the cytoplasm and contained with lysosomal structures. x 8 000. Fig. 7. Six-day vitamin A-treated explant. A higher magnification of a widened intercellular space showing microvilli with an associated filamentous surface coat. Tonofilaments are present in small bundles but are rarely seen close to the cell periphery. Desmosomes appear to be breaking down. The overall structure is reminiscent of a canaliculus. x 39OOG. Fig. 8. Six-day vitamin A-treated explant. An intercellular junction resembling a tight junction (0) has appeared at the periphery of a widened intercellular space. Projecting microvilli with a filamentous coating are prominent. x70000. Fig. 9. Six-day vitamin A-treated explant. Cells b and c appear to be undergoing a morphogenetic change in response to vitamin A, whereas cell a is not responding. Remnants of desmosomal plaques (dp) which presumably were formerly apposed can be identified. Incell c there is a progressive increase in the extent of microvillus development. A filamentous surface coat is present on the microvilli of cells b and c but is not present on the surface of cell a. The remaining desmosomes appear to be breaking down. x 39 000. Exptl Cell Res 76 (1973)
In vitro effect of vitamin A on epithelium 121
Exptl Cell Res 76 (1973)
122 M. L. Barnett & G. Szabo
Exptl Cell Res 76 (1973)
In vitro effect of vitamin A on epithelium
123
Exptl Cell Res 76 (1973)
124 M. L. Barnett & G. Szabo
Exptl Cell Res 76 (1973)
In vitro effect of vitamin A on epithelium junctional complexes resembling tight junctions (zonula occludens) [18] could be identified, thus enhancing the resemblance to a glandular structure (fig. 8). Other changes which were seen included an apparent increase in extent of Golgi regions, and small vesicles, found both within the cytoplasm and in association with the cell membrane. No membrane coating granules were present. One section (fig. 9) was particularly interesting because it suggested a sequence of events which may result in the characteristic surface changes. Three epithelial cells are shown, two of which appear to be responding to vitamin A, whereas the uppermost one appears not to have responded. Remnants of formerly apposing desmosomal plaques can be seen in all cells. However, in the responding cells microvilli, with a filamentous surface coat, show progressively better formation as they get farther away from the desmosomal remains. This suggests that an initial step in the morphogenetic change is a destruction of desmosomes and, in responding cells, attachment plaques as well, with subsequent retraction and loss of tonofilaments and microvillus formation. As desmosomes are lost, intercellular spaces widen. Other changes, including the appearance of ‘tight junctions may be simultaneous with, or subsequent to, these initial cellular alterations. DISCUSSION The morphologic changes described above and the absence of any signs of keratinization in the treated explants indicate that vitamin A can, indeed, influence the morphogenesis of adult mammalian epidermis in vitro. Our findings are consistent in many respects with those of a previous ultrastructural study on altered embryonic chick skin [4]. In particular, the findings of a decrease
125
in the size of tonofilament bundles, the formation of intercellular lacunae and microvilli, and a general decrease in cell density have been mentioned as being characteristic of transformed chick skin [4]. In addition, mucus globules were described in treated chick epidermis [4]. Whereas such globules were not seen in our sections, the formation of microvilli containing a well developed surface coating and the appearance of tightjunction-like complexes indicates that the tissue has undergone a significant transformation. Of perhaps greater interest is a comparison of the findings described herein with those from a study on in vivo squamous metaplasia of adult tracheal epithelium in vitamin Adeficient rats [19]. Our epithelial cells, transforming from a keratinizing type to a mucous type, share a great many features with tracheal cells which are midway along the path of squamous metaplasia from a respiratory type. These results, in combination with past findings, suggest that adult mammalian epithelia may show a continuum of morphogenetic responses to vitamin A, depending upon the threshold of a given epithelium
[l, 201. Whereas vitamin A per se may not be involved in an inductive interaction between adult dermis and epidermis in vivo, the fact that adult epithelium exposed to an exogenous agent can undergo the marked changes described above also lends indirect support to the theory for the conservation of epithelia1 specificity which states that the pathway of differentiation in any given region followed by presumably equipotential germinative epithelial cells is dependent upon a specific inductive stimulus from the underlying dermis
1211. Although early changes in vitamin Atreated cells appear to involve desmosomal Exptl Cell Res 76 (1973)
126 M. L. Barnett and G. Szabo complexes, the exact mechanism of action of the vitamin in producing this change is unknown. In view of the widespread changes seen in the treated tissue, it does not seem unreasonable to assume that the agent in some way ultimately affects gene expression [22,23]. Certainly, several possibilities exist by which this can occur, either directly or indirectly. However, the mechanism by which epithelial morphogenesis is affected remains to be elucidated. This study was supported by USPHS Grants DE00111 and DE-01766 from the National Institute of Dental Research.
REFERENCES 1. Fell, H B & Mellanby, E, J physiol 119 (1953) 470. 2. Fell, H B, Proc roy sot London, ser B 146 (1957) 242. 3. Pelt, S R & Fell, H B, Exptl cell res 19 (1960) 99.
4. Fitton Jackson, S & Fell, H B, Dev biol 7 (1963)
7. Bern, H A, Elias, J J, Pickett, P B, Powers, T R & Harkness, M N, Am j anat 96 (1955) 419. 8. Lawrence, D L & Bern, H A, J invest derm 31 (1958) 313. 9. Sabella, J D, Bern, H A & Kahn, R H, Proc sot exptl biol med 76 (1951) 499. 10. New, D A T, Brit j derm 75 (1963) 320. 11. - Exptl cell res 39 (1965) 178. 12. Szabo, G, Fundamentals of keratinization (ed E 0 Butcher & R Sognnaes) D. 45. Amer assoc advancement sci, Waihington-(1962). 13. Biggers, J D, Gwatkin, R B L & Heyner, S, Exptl cell res 41 (1961) 24. 14. Ito, S & Karnovsky, M, J cell biol 39 (1968) 168a. 15. Luft, J H, J biophys biochem cytol 9 (1961) 409. 16. Reynolds, E S, J cell biol 17 (1963) 208. 17. Ito, S, J cell biol 27 (1965) 475. 18. Farquhar, M G & Palade, G E, J cell biol 17 (1963) 375. 19. Wong, Y-C & Buck, R C, Lab invest 24 (1971) 55.
20. Wilde, C E, Cell, organism and milieu (ed D Rudnick) n. 3. The Ronald Press, New York (1959). ’ 21. Billingham, R E & Silvers, W K, J exptl med 125 (1967) 429. 22. deluca, L &Wolf,
G, Am j clin nutr 22 (1969) 1050. 23. Prutkin, L, J invest derm 57 (1971) 323.
394.
5. Rothberg, S, J invest derm 49 (1967) 35. 6. Montagna, W, Proc sot exptl biol med 86 (1954) 668.
Exptl Cell Res 76 (1973)
Received May 23, 1972 Revised version received July 21, 1972