Ciliated and Secretory Epidermis Produced from Emb ryo n ic Mammal ian Skin in Organ Culture by Vitamin A PHILLIP R. SWEENY AND MARGARET H. HARDY Departments of Microbiology and Biomedical Sciences, University of Guelph, G u e i p k , Ontario, Canada N l G 2W2

ABSTRACT Whcn skin from the upper lip of 12-day embryonic mice was grown for ten days in organ culture with 5.7 pug retinol added per ml of biological medium, keratinization was suppressed and a ciliated and secretory epithelium was produced. Ultrastructural features of this epithelium are described. At this very early stage mouse epidermis is thus similar to chick epidermis in its ability to undergo radical metaplasia in response to vitamin A.

Vitamin A (retinol) in high doses can transform early embryonic chick epidermis into a ciliated, mucus-secreting epithelium, thereby demonstrating that the skin of 7to 12-day old chick embryos is not irreversibly committed to keratinization (Fell and Mellanby, ’53; Fell, ’57). Many attempts to produce similar changes in pre-natal or adult mammalian skin by vitamin A have been unsuccessful, although less extreme changes have been produced by vitamin A in several species of rodents (New, ’65; Hardy, ’67; Barnett and Szabo, ’73). Only the lining of the hamster cheek pouch, which is not skin, has become mucussecreting (Lawrence and Bern, ’60). We have completed an extensive study of the ultrastructural changes produced when 14-day embryonic mouse skin was grown in organ culture with excess vitamin A (Hardy, Sweeny and Bellows, ’76, in preparation). Changes in dermis and epidermis were found to precede the vitamin A-induced modifications of still keratinizing epidermis which have been described as “partial metaplasia” (Hardy, ’67). Our purpose in this brief communication is to report that when a few pieces of even younger skin (12 days) were cultivated for ten days with excess vitamin A, keratinization was suppressed completely, or over more than half the surface, and the epithelium became ciliated and secretory, a5 in the chick embryo skin thus treated. The latter condition will be described as “complete metaplasia.” No previous report ANAT. REC., 785: 93-100.

of a ciliated epithelium obtained by transformation of mamalian epidermis by any agent in vitro has been noted. MATERIALS AND METHODS

Pieces of skin from the upper lip of Swiss albino mouse embryos at 12 days of gestation were grown on 3 : 1 cock plasma and chicken embryo extract clots in Maximow slide preparations as described previously (Hardy, ’67). Care was taken during dissection to exclude the adjacent buccal epithelium and nasal epithelium. The standard medium contained 4.5 J ethanol/ ml, and vitamin A medium contained 6 retinol in 4.5 ,1 ethano1,’ml. The progress of the explants was observed daily under the microscope, Explants were fixed in toto after 10 days in 3 % glutaraldehyde in isotonic Sorenson’s phosphate buffer at pH 7.3, then post-fixed in 1% osmium in phosphate buffer and embedded in Epon (Luft, ’61). Sections at 1-2 were cut through the whole explants with a PorterBlum Ultramicrotome 11. Sample sections were mounted and examined by phase contrast microscopy until the metaplastic epidermis or other structure of interest was cut at a suitable level. Ultra-thin sections were immediately cut, and collected, unsupported, on 200 mesh copper grids. Another semi-thin section was then collected and the procedure continued until the tissue was cut right through, supplying from 3-10 sets of ultra-thin sections at Received July 10, ’75. Accepted Dec. 9, ’75.

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different levels through the explant, and from 10-30 semi-thin sections. The latter were stained with 1% methylene blue in 1% borax (Jeon, '65) to provide a reference set of sections for light microscopy, The semi-thin sections adjacent to the ultra-thin ones were photographed and used to locate areas and cells for ultra-structural study. The ultra-thin sections were stained with uranyl acetate followed with Millonig's lead citrate (Millonig, '61) and examined in a Phillips 300 electron microscope. RESULTS

One explant from a 12-day old embryo grown in the standard medium rolled into a ball with the epidermal surface inwards, as is usual with skin from 14-day old or younger embryos in this type of culture. The epidermis stratified as in vivo, and keratinized cells were recognized in the living explant at the epidermal surface after five days in vitro. Hair follicles developed normally, and hairs were emerging through the keratinized epidermis after nine days in vitro. Semi-thin sections of this explant after ten days revealed a normal keratinized epidermis (fig. I), and ultra-thin sections confirmed the presence of tonofilaments, tonofibrils, keratohyalin and keratin. Squamous stratification and keratinization were not observed at any time in the corresponding explant grown in the vitamin A medium, and the epithelial outgrowths which initially resembled early hair follicles did not continue to develop in this direction. The epidermis rolled up and enlarged but the cells remained polyhedral. Semi-thin sections showed a large central space filled with apparently shed epithelial cells (fig. 2 ) . Next to the central area was a stratified cuboidd type of epithelium containing numerous superficial ciliated cells (fig. 3). Within the epithelium small cavities and channels were observed (fig. 2 ) . In the electron microscope, the cells lining the central lumen were found to be joined by junctional complexes. Some cells bore both cilia and filopodia, while others had only large numbers of filopodia of uniform thickness which looked like elongated microvilli (fig. 4). Many of the smaller

channels and spaces were also filled with cilia and,, or microvilli. The cilia were present in very large numbers in any one cell, and showed typical microtubular arrays, as well as basal bodies (fig. 5). In another experiment, a pair of explants from a 12-day old embryo also showed marked differences due to the medium. In standard medium, keratinization of the epidermis was observed at 5 days, and, at 7 days, keratinized hairs, which emerged at 9 days. In vitamin A-enriched medium, keratinization began at one end of the explant after seven days but more than half of the epidermis remained unstratified even after ten days. Epithelial outgrowths at the keratinizing end did not develop beyond the earliest stages of hair follicles, and epithelial outgrowths at the non-keratinized end developed abnormally. After ten days the explants were fixed, and the above observations were confirmed by light and electron microscopy, The structure of the unkeratinized half of the epidermis grown with vitamin A resembled that of the first explant grown in the presence of the vitamin. Small lumina with microvilli, and smaller channels were seen. The second explant showed fewer cilia but very numerous filopodia on the cuboidal cells lining the internal channels. In both the vitamin A-treated explants most of the microvilli and filopodia were surrounded by a layer of fuzzy material similar to the glycocalyx of intestinal microvilli and other transporting epithelia (Berridge and Oschman, '72). Frequently the internal channels were filled with fuzzy matcrial of similar intermediate electron density (fig. 6 ) and sometimes filamentous material was seen to radiate from microvilli (fig. 7 ) . Large amorphous masses with higher electron density were also seen in these channels (fig. 6). The cells lining the channels were well supplied with prominent Golgi apparatus, an image not reported in supra-basal cells of a normally keratinizing epidermis. DISCUSSION

In a light microscopic histochemical study of 13-day mouse embryonic skin from the upper lip in organ culture (Bellows and Hardy, '76, submitted for publication), the effects of excess vitamin A

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were found to be intermediate between the both authors from the National Research partial metaplasia observed in 14-day skin Council of Canada. and the complete metaplasia reported LITERATURE CITED above in 12-day skin. In 13-day skin after 11 to 21 days a small proportion of the Barnet, M. L., and G. Szaho 1973 Effect of vitamin A on epithelial morphogenesis in vitro. epidermis was non-keratinizing and seFine structural changes i n explants of adult creted a PAS-positive, diastase-resistant, mammalian skin, Exptl Cell Res., 76: 118-126. Alcian blue-negative or feebly positive ma- Bellows and Hardy (1976, submitted for publiterial. Further histocheinical analysis has cation) Histochemical evidence of mucosubsuggested that either non-sulfated acidic stances i n the metaplastic epidermis and hair follicles produced i n vitro i n the presence of exmucosubstances such as sialomucins, or cess vitamin A. neutral mucosubstances were present. Berridge, M. J., and J. L. Oschman 1972 TransThus it seems likely that the fuzzy and porting Epithelia. Academic Press. New York. amorphous material reported above in the Fell, H. B. 1957 The effect of excess vitamin A on cultures of embryonic chicken skin excorresponding areas of 12-day embryonic planted at different stages of differentiation. skin cultivated for ten days in excess vitaProc. Roy. Sac. (London), Ser. B.. 146: 242-256. min A contained similar mucosubstances. Fell, H. B., and E. Mellanbp 1953 Metaplasia Proof of this is being sought by electron produced i n cultures of chick ectoderni by high vitamin A. J. Physiol., (London). 119: 470-488. microscopic histochemistry. What is the significance of finding cilia Hardy, M. H. 1967 Responses i n embryonic mouse skin to excess vitamin A in organotypic in 12-day embryonic mouse skin after ten cultures from the trunk, upper lip and lower days of treatment with vitamin A? Our jaw. Exptl Cell Res.. 46: 367-384. interpretation is that at 12 days the epi- Hardy, Sweeny and Bellows (1976. in preparation) The effect of vitamin A o n foetal mouse dermis of this mammal, like that of the 7epidermis in organ culture - a n ultrastructural to 12-day chick, is in the protodifferentistudy. ated state (Rutter et al., '68; Hay, '74), Hay, E. D. 1974 Cellular basis of regeneration. and may respond to environmental influIn: Concepts of Development. J . Lash and J . R. Whittaker, eds. Sinauer, Stanford, pp. 404-428. ences by selecting from among a large repertoire of phenotypic expressions. At 13 Jeon, K. W. 1965 Simple method for staining and preserving epoxy resin embedded animal days the epidermal cells sometimes retissue sections for light microscopy. Life Scisponded to vitamin A by mucous metaences, 4: 1839-1841. plasia although up to the present cilia have Lawrence, D. J., and H. A. Bern 1960 Mucous metaplasia and mucous gland formation i n not been observed, and at 1 4 days the keratinized adult epithelium in sitti treated usual response was keratinization with with vitamin A. Exptl Cell Res., 21: 443-446. some additional production of mucosub- Luft, J. H. 1961 Improvements i n epoxy resin stances. A wide range of dose levels proembedding methods. J. Biophys. Biochem. Cytol., 9: 409-414. duced similar results at the same age G. 1961 A modified procedure for (Hardy, '67; and unpublished data). In the Millonig, lead staining of thin sections. J. Biophys. Biochick also the range of response became chem. Cytol., 1 1 : 736-739. more restricted as epidermal differentia- New, D. A . T. 1965 Effects of excess vitamin A on cultures of skin from the tail and pads tion proceeded (Fell, '57). That the ability of the embryonic rat, and from the trunk, tail of the epidermis to respond to vitamin ,4 and pads of the embryonic rabbit. Exptl Cell can be correlated with its initial ultrastrucRes., 39: 178-183. ture will be shown in the full-length paper Rutter, W. J., 1%'. R. Clark, J. D. Kemp, W. S. Bradshaw, T. G . Saunders and W. D. Ball in preparation. ACKNOWLEDGMENTS

This study was supported by grants to

1968 Multiphasic regulation i n cytodifferentiation. I n : Epithelial-Mesenchymal Interactions. R. Fleishmajer and R. E. Billingham, eds. Williams and Wilkins, Baltimore, pp. 114-131.

PLATE 1 EXPLANATION OF FIGURES

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1

Semi-thin section of skin from 12-day embryo after ten days i n standard medium, showing stratified keratinized epidermis diflerentiated in vitro. Note the dark granules of keratohyalin i n the stratum granulosum and the dark superficial flattened cells of the stratum corneum. x 649.

2

Semi-thin section of skin from 12-day embryo after 10 days i n vitam i n A-enriched medium. Epidermal cells did not become squamous or develop granules, but retained a polyhedral form. T h e innermost cells of the epithelium line a large central cavity containing cells with pyknotic nuclei. Small internal channels are seen. 'i649.

3

Cuboidal epithelial cells lining the central cavity i n a n area adjacent to that shown in figure 2. Numerous cells display cilia. Nomarski interference photograph. A 2,363.

4

Electron micrograph from the vitamin A-treated explant shown in figures 2 and 3 showing apices of three epithelial cells lining a n internal cavity. Note the junctional complexes between cells at the luminal border. The cell i n the lower center bears microvilli and numeruus cilia with basal bodies, while the cell on the right is rich 40,500. i n glycogen and bears microvilli.

CILIATED EPIDERMIS FROM MOUSE SKIN I N VITRO WITH VITAMIN A Phillip R . Sweeny and Margaret H . Hardy

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PLATE 2 EXPLANATION OF F I G U R E S

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5

Electron micrograph of cilia and microvilli from the vitamin A-treated explant illustrated in figures 2-4. Two cross-sections of cilia showing typical microtubular arrays are seen at the lower left and a longitudinal section of cilium with microtubules near the center. Basal bodies are at the right. x 60,750.

6

Electron micrograph of a small lumen lined with microvilli in the epithelium of a vitamin A-treated explant. The lumen contains finely granular material and a single mass of material of greater electron density. Electron-dense material also appears to coat the surface of cells and microvilli. x 44,625.

7

Electron micrograph of the apices of two cells bearing microvilli. Filamentous materials appear to radiate from the surface of microvilli of the cell on the left. The membrane on the right has a more dense surface coat and the intervillous material appears more electron dense. x 52,650.

CILIATED EPIDERMIS FROM MOUSE SKIN I N VITRO WITH VITAMIN A Phillip R. Sweeny and Margaret H . Hardy

PLATE 2

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Ciliated and secretory epidermis produced from embryonic mammalian skin in organ culture by vitamin A.

When skin from the upper lip of 12-day embryonic mice was grown for ten days in organ culture with 5.7 mug retinol added per ml of biological medium, ...
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