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On the nature of tissue interactions in embryonic skin
233 ON THE
NATURE
OF TISSUE
INTERACTIONS
IN EMBRYONIC
SKIN
J. W. DODSON Strangeways
Research Laboratory,
Cambridge,
England
Received April 12, 1963
s
TUDIES of the interactions between epithelia and mesenchyme in developing embryonic organs have shown that mesenchyme has an important influence on epithelial growth and differentiation in, for example, salivary gland [5], pancreas [3], feather germs [ill, and preen gland [4]. In skin, McLaughlin [8, 91 and more recently (since this investigation began) Wessells [13] have found that the proximity of mesenchyme is necessary for the continued survival of epidermis, and for its normal differentiation. The mechanism of this dependence is less clear, but the participation of the intercellular material in such interactions has frequently been suggested (e.g. [6, 7, 91). In order fully to understand the relationship between tissues, it is necessary to know which components of one tissue contribute to the continued growth, differentiation, and functioning of the other. The work briefly reported here is an attempt to analyse the relationship between embryonic epidermis and dermis, in order to determine which mesenchymal constituents affect epidermal survival, differentiation and keratinization. The experiments were made on the scaly skin of the anterior tarsometatarsal region of the 12-day embryonic chick. After the skin had been treated with 0.04 per cent Versene (ethylene diamine tetra-acetic acid, di-sodium salt) for 20 min at 20°C the epidermis could be easily and cleanly peeled from the dermis. The dermis was subjected to various experimental treatments, and the two tissues were then recombined and cultured in vitro on cellulose acetate rafts [IO] on clots made of plasma and embryo extract [2]. The explants were fixed in acetic-Zenker for histological examination. The epidermis at 12 days consists of a layer of columnar basal cells and a single stratum of intermediate cells, above which is a two layered periderm. After separation and recombination with epidermal basal cells next to the dermis, both dermis and epidermis grew well, the periderm differentiated rapidly then degenerated, and layers of birefringent keratin were produced (Fig. 1). Isolated epidermis, however, did not survive under these conditions; the basal cells lost their columnar orientation within 3 hr, the epidermal sheet contracted and thickened, and the organization of cell layers was lost. Mitosis ceased after 18-24 hr, and most of the lower cells became necrotic, only a few cells showing traces of keratin, although the periderm underwent some differentiation (Fig. 2). No basement membrane formed. This behaviour occurred when isolated epidermis was placed on rafts, on millipore filters, or directly on the clot. Dermis was treated with trypsin to obtain a suspension of cells almost free of dermal intercellular material. When this suspension was placed on the basal surface of the epidermis, the orientation of the basal cells and the organization of cell layers were maintained, and a basement membrane stainable with the periodic acid-Schiff
Experimenlal
Cell Research 31
Fig. l.-Epidermis Fig. 2.-Isolated
recombined epidermis
with
untreated,
live
grown on a rayon-acetate
Fig. S.-Epidermis
recombined
Fig. 4.-Epidermis
grown on a
with
frozen-killed
dermis. raft.
dermis.
10 days in culture.
&an.
x200.
2 days in culture.
Azan.
x 400.
10 days in culture.
Azan.
x 200.
colfagen gel. 10 days in culture. Azan. x 200.
(PAS) technique, was formed within 3 hr. The dermal cells synthesized intercellular materials and reconstituted a dermis of almost normal appearance, while the epidermis continued to differentiate and keratinize. When a similar suspension of cells was placed on the peridermal surface, however, there was no contact between the tissues, and the epidermis behaved as though isolated, although the dermal cells again reconstituted a dermis. Dermis was killed by repeated freezing and thawing, in order to leave the intercellular material relatively unaltered. The remains of cells were present, but no living cells were found in this material after culturing. Epidermis recombined with this frozen-killed dermis maintained the basal cell orientation, and a basement membrane formed, again within 3 hr. The epidermis spread over the dermis, maintained its organization, and continued to survive, differentiate, and produce layers of keratin for at least 10 days. Prominent features in these recombinations were the presence within the dermis of intrusive spurs of basal epidermal cells, and a reorientation of dermal fibres from being parallel to the epidermal surface to being perpendicular to it (Fig. 3). Similar frozen-killed dermis was treated with trypsin, and used in recombinations. The orientation of the epidermal basal cells was maintained, and a basement membrane formed, but the epidermis did not spread and there were many epidermal spurs in the dermis. The epidermis produced layers of keratin, but did not survive long; by 5 days in vitro most of the epithelium was keratinized, leaving only detached, non-viable basal cells. Dermis was also killed by heat; epidermis placed on this behaved like isolated epidermis. Experimenful
Cell Research 31
235
Tissue interactions in skin
Since collagen is one of the main constitutents of the dermis, its importance to epidermis was assessed by using a gel of collagen as a substitute for dermis. The gels of acetic acid-extracted collagen were made by Simkovic’s modification [12] of Ehrman and Gey’s method [l], but they were sterilized by ultraviolet irradiation, and were not dried. Epidermis placed on these gels survived, with cuboidal basal cells, progressive differentiation and the production of layers of birefringent keratin, for at least 10 days (Fig. 4). The epidermis spread, and the gel was seen to be contracted. On smooth-surfaced gels there were no epidermal spurs within the gel. PAS-staining material was present under the basal epidermal cells in some cultures, but not in all. It would appear, therefore, that, under these conditions, collagen alone is sufficient for epidermal survival, but in these collagen gels small amounts of other proteins and polysaccharides might have been present, and these may also be important. The changes in the epidermis after combination with dermis exposed to tryptic digestion or heat treatment suggest that other dermal components besides collagen may be important to the epidermis, and it is hoped that further experiments will enable these substances to be identified. From these results it is concluded that: (a) the survival of 12-day embryonic chick epidermis as an organized, keratinizing tissue depends on the contact of the basal surface with a suitable substratum; (b) the presence of dermal cells is not essential for adhesion to the substratum or for survival and keratinization of the epidermis; (c) trypsin-labile and heat-labile components of the dermis are important for these processes; (d) collagen plays an important role in the provision of such a substratum; (e) the epidermis may influence the orientation of dermal fibres, possibly by the exertion of tensile forces; (f) the formation of a PAS-staining basement membrane can occur in the absence of living dermal cells. I wish to express my sincere thanks to Dr. H. B. Fell, F.R.S., and to Dr. S. Fitton Jackson for many helpful discussions and stimulating ideas, and I am indebted to the Medical Research Council for the receipt of a Research Scholarship. REFERENCES 1. 2. 3. 4. 5. A. 7. 8.
9. 10.
11. 12. 13.
EHRMAS, R. L. and GEY, G. O., J. iVat[. Cancer Insf. 16, 1375 (1956). FELL, H. B. and ROBINSON, R., Biochem. J. 23, 767 (1929). GOLOSOW, N. and GROBSTEIN, C., Deuefop. Biof.4, 242 (1962). GOMOT, L., J. Embryof. Expff. Morphof. 6, 162 (1958). GROBSTEIN, C., J. Exptl. Zool. 124, 383 (1953). GROBSTEIN, C., Advances in Cancer Research 4, 187 (1956). b'IOSCONA, A., .I. Cellular Comp. Physiol. 60, Suppl. 1, 65 (1962). BmOUGHLIS, C. B., J. Embryol. Exptf. Morphol. 9, 370 (1961). -ibid. 9, 385 (1961). SCHAFFER, B. M., Expff. Cell Res. 11, 244 (1956). SENGEL, P., Ann. sci. nut. Zool. et biof. animaIe (11) 20, 431 (1958). SIMKOVIC, I)., Expff. Cell Res. 17, 573 (1959). \\‘ESSELLS, N. I<., Deuefop. Biof. 4, 87 (1962).
Experimenfal
Cell Research 31
NATURE
OF TISSUE
INTERACTIONS
IN EMBRYONIC
SKIN
J. W. DODSON Strangeways
Research Laboratory,
Cambridge,
England
Received April 12, 1963
s
TUDIES of the interactions between epithelia and mesenchyme in developing embryonic organs have shown that mesenchyme has an important influence on epithelial growth and differentiation in, for example, salivary gland [5], pancreas [3], feather germs [ill, and preen gland [4]. In skin, McLaughlin [8, 91 and more recently (since this investigation began) Wessells [13] have found that the proximity of mesenchyme is necessary for the continued survival of epidermis, and for its normal differentiation. The mechanism of this dependence is less clear, but the participation of the intercellular material in such interactions has frequently been suggested (e.g. [6, 7, 91). In order fully to understand the relationship between tissues, it is necessary to know which components of one tissue contribute to the continued growth, differentiation, and functioning of the other. The work briefly reported here is an attempt to analyse the relationship between embryonic epidermis and dermis, in order to determine which mesenchymal constituents affect epidermal survival, differentiation and keratinization. The experiments were made on the scaly skin of the anterior tarsometatarsal region of the 12-day embryonic chick. After the skin had been treated with 0.04 per cent Versene (ethylene diamine tetra-acetic acid, di-sodium salt) for 20 min at 20°C the epidermis could be easily and cleanly peeled from the dermis. The dermis was subjected to various experimental treatments, and the two tissues were then recombined and cultured in vitro on cellulose acetate rafts [IO] on clots made of plasma and embryo extract [2]. The explants were fixed in acetic-Zenker for histological examination. The epidermis at 12 days consists of a layer of columnar basal cells and a single stratum of intermediate cells, above which is a two layered periderm. After separation and recombination with epidermal basal cells next to the dermis, both dermis and epidermis grew well, the periderm differentiated rapidly then degenerated, and layers of birefringent keratin were produced (Fig. 1). Isolated epidermis, however, did not survive under these conditions; the basal cells lost their columnar orientation within 3 hr, the epidermal sheet contracted and thickened, and the organization of cell layers was lost. Mitosis ceased after 18-24 hr, and most of the lower cells became necrotic, only a few cells showing traces of keratin, although the periderm underwent some differentiation (Fig. 2). No basement membrane formed. This behaviour occurred when isolated epidermis was placed on rafts, on millipore filters, or directly on the clot. Dermis was treated with trypsin to obtain a suspension of cells almost free of dermal intercellular material. When this suspension was placed on the basal surface of the epidermis, the orientation of the basal cells and the organization of cell layers were maintained, and a basement membrane stainable with the periodic acid-Schiff
Experimenlal
Cell Research 31
Fig. l.-Epidermis Fig. 2.-Isolated
recombined epidermis
with
untreated,
live
grown on a rayon-acetate
Fig. S.-Epidermis
recombined
Fig. 4.-Epidermis
grown on a
with
frozen-killed
dermis. raft.
dermis.
10 days in culture.
&an.
x200.
2 days in culture.
Azan.
x 400.
10 days in culture.
Azan.
x 200.
colfagen gel. 10 days in culture. Azan. x 200.
(PAS) technique, was formed within 3 hr. The dermal cells synthesized intercellular materials and reconstituted a dermis of almost normal appearance, while the epidermis continued to differentiate and keratinize. When a similar suspension of cells was placed on the peridermal surface, however, there was no contact between the tissues, and the epidermis behaved as though isolated, although the dermal cells again reconstituted a dermis. Dermis was killed by repeated freezing and thawing, in order to leave the intercellular material relatively unaltered. The remains of cells were present, but no living cells were found in this material after culturing. Epidermis recombined with this frozen-killed dermis maintained the basal cell orientation, and a basement membrane formed, again within 3 hr. The epidermis spread over the dermis, maintained its organization, and continued to survive, differentiate, and produce layers of keratin for at least 10 days. Prominent features in these recombinations were the presence within the dermis of intrusive spurs of basal epidermal cells, and a reorientation of dermal fibres from being parallel to the epidermal surface to being perpendicular to it (Fig. 3). Similar frozen-killed dermis was treated with trypsin, and used in recombinations. The orientation of the epidermal basal cells was maintained, and a basement membrane formed, but the epidermis did not spread and there were many epidermal spurs in the dermis. The epidermis produced layers of keratin, but did not survive long; by 5 days in vitro most of the epithelium was keratinized, leaving only detached, non-viable basal cells. Dermis was also killed by heat; epidermis placed on this behaved like isolated epidermis. Experimenful
Cell Research 31
235
Tissue interactions in skin
Since collagen is one of the main constitutents of the dermis, its importance to epidermis was assessed by using a gel of collagen as a substitute for dermis. The gels of acetic acid-extracted collagen were made by Simkovic’s modification [12] of Ehrman and Gey’s method [l], but they were sterilized by ultraviolet irradiation, and were not dried. Epidermis placed on these gels survived, with cuboidal basal cells, progressive differentiation and the production of layers of birefringent keratin, for at least 10 days (Fig. 4). The epidermis spread, and the gel was seen to be contracted. On smooth-surfaced gels there were no epidermal spurs within the gel. PAS-staining material was present under the basal epidermal cells in some cultures, but not in all. It would appear, therefore, that, under these conditions, collagen alone is sufficient for epidermal survival, but in these collagen gels small amounts of other proteins and polysaccharides might have been present, and these may also be important. The changes in the epidermis after combination with dermis exposed to tryptic digestion or heat treatment suggest that other dermal components besides collagen may be important to the epidermis, and it is hoped that further experiments will enable these substances to be identified. From these results it is concluded that: (a) the survival of 12-day embryonic chick epidermis as an organized, keratinizing tissue depends on the contact of the basal surface with a suitable substratum; (b) the presence of dermal cells is not essential for adhesion to the substratum or for survival and keratinization of the epidermis; (c) trypsin-labile and heat-labile components of the dermis are important for these processes; (d) collagen plays an important role in the provision of such a substratum; (e) the epidermis may influence the orientation of dermal fibres, possibly by the exertion of tensile forces; (f) the formation of a PAS-staining basement membrane can occur in the absence of living dermal cells. I wish to express my sincere thanks to Dr. H. B. Fell, F.R.S., and to Dr. S. Fitton Jackson for many helpful discussions and stimulating ideas, and I am indebted to the Medical Research Council for the receipt of a Research Scholarship. REFERENCES 1. 2. 3. 4. 5. A. 7. 8.
9. 10.
11. 12. 13.
EHRMAS, R. L. and GEY, G. O., J. iVat[. Cancer Insf. 16, 1375 (1956). FELL, H. B. and ROBINSON, R., Biochem. J. 23, 767 (1929). GOLOSOW, N. and GROBSTEIN, C., Deuefop. Biof.4, 242 (1962). GOMOT, L., J. Embryof. Expff. Morphof. 6, 162 (1958). GROBSTEIN, C., J. Exptl. Zool. 124, 383 (1953). GROBSTEIN, C., Advances in Cancer Research 4, 187 (1956). b'IOSCONA, A., .I. Cellular Comp. Physiol. 60, Suppl. 1, 65 (1962). BmOUGHLIS, C. B., J. Embryol. Exptf. Morphol. 9, 370 (1961). -ibid. 9, 385 (1961). SCHAFFER, B. M., Expff. Cell Res. 11, 244 (1956). SENGEL, P., Ann. sci. nut. Zool. et biof. animaIe (11) 20, 431 (1958). SIMKOVIC, I)., Expff. Cell Res. 17, 573 (1959). \\‘ESSELLS, N. I<., Deuefop. Biof. 4, 87 (1962).
Experimenfal
Cell Research 31