The Differentiation of Epidermis II

/. Embryol. exp. Morph., Vol. 17, 1, pp. 107-117, February 1967 107 With 4 plates Printed in Great Britain

The differentiation of epidermis II. Alternative pathways of differentiation of embryonic chicken epidermis in organ culture

ByJ. W. DODSON1 From the Strangeways Research Laboratory, Cambridge

INTRODUCTION In the present study, two series of experiments have been made to investigate the role of the dermis in determining alternative pathways of differentiation in the epidermis. Previous work has shown that the scaly metatarsal epidermis of 12-day chicken embryos, when isolated in culture on a plasma clot or various other substrata, fails to develop normally and undergoes a characteristic sequence of differentiative and degenerative changes (McLoughlin, 1961a; Wessells, 1962;Dodson, 1963,1966). On the other hand, the separated epidermis, when cultivated in combination with either its own dermis or a gel of collagen, survives and forms a stratum corneum (Dodson, 1963, 1966). The questions arise of whether the changes produced in the epidermis by growth in isolation can be reversed by subsequent recombination of the epithelium with the dermis, and if so at what stage the degeneration becomes irreversible. Accordingly, in the first series of experiments, epidermis was cultivated in isolation for various periods of time, then recombined with dermis and cultivated further; the explants were examined histologically for signs of a keratinizing epithelium. In a similar experiment, Wessells (1963) found that proliferating columnar basal cells appeared in epidermis isolated for 10 or 24 h, then recombined with dermis for a further 1 or 2 days, but longer periods of segregation were not tested, nor was further differentiation described. The differentiation of the epidermis of embryonic chicken skin in culture can also be altered profoundly by treatment with excess of vitamin A (Fell & Mellanby, 1953; Fell, 1957). When the whole skin of 7-, 13-, or 18-day embryos was treated, keratinization was inhibited and a mucous metaplasia was induced; the latter change, however, was less extensive and less frequent in the oldest skin. Originally it was not known whether this action of the vitamin was directly on the epidermis or whether it was mediated through changes produced in the dermis. McLoughlin (1961a) noted, however, that in isolated limb 1 Author's address: Department of Zoology, University of Bristol, Bristol, U.K. 108 J. W. DODSON epidermis from 5-day chicken embryos, squamous changes associated with slight keratinization in control explants were inhibited by the vitamin and in the treated explants a thin layer of mucus appeared; this result indicated that in this very young material the changes were due to a primary action of the vitamin on the epithelium. The second series of experiments recorded in the present paper was made to determine whether the vitamin also acts directly on the partly differentiated epidermis of older embryos and whether the isolated epithelium from such embryos can undergo a full mucous metaplasia when grown on a collagen gel in the presence of excess of vitamin A.

MATERIALS AND METHODS Epidermis was separated from dermis after Versene treatment of the scaly skin of the anterior tarso-metatarsal region of 12-day chicken embryos (stages 37^-38^ of Hamburger & Hamilton, 1951). The techniques of separation, cultivation, and histological examination have been described in a previous paper (Dodson, 1966). Sheets of separated epidermis were spread over either rayon-acetate rafts (Schaflfer, 1956) or pieces of Millipore filter, type HA (Millipore Filter Corp., Bedford, Mass.); they were then cultivated for 12-48 h at 37-5 °C by the watch-glass technique of Fell & Robison (1929) on clots made of 12 drops of fowl plasma and 8 drops of embryo extract. The pieces of separated dermis were placed on rayon rafts on clots and kept at room temperature (18-20 °C) until required for recombination, except that when epidermis was isolated for 48 h, dermis was prepared freshly immediately before recombination. After incubation the epidermis was carefully removed from the raft or filter and was placed on the inner surface of a piece of dermis (i.e. the surface not bearing the basement membrane) on a raft; a second piece of dermis was then placed over the epithelium, again with its inner surface next to the latter tissue. This 'sandwich' technique obviated the necessity of determining the basal surface of the twisted sheet of isolated epidermis, and placing the inner surface of the dermis next to the epithelium prevented the extant basement membrane on the dermis from being confused with any newly formed basement membrane next to the epidermis. The recombined tissues were cultivated on a clot as before, for periods ranging from 3 h to 7 days. For the experiments on the effect of vitamin A, gels of collagen from acetic acid-extracted rat tail tendon were prepared as described in the previous paper. Vitamin A alcohol was dissolved in ethanol and added to fresh fowl plasma to give a concentration of either 2-4 or 9 i.u. per ml in the final plasma-embryo extract clot. The medium for control explants contained the same amount of ethanol (0-1 %). Pieces of collagen gel were soaked in clot exudate (H. B. Fell, personal communication) containing either vitamin A or ethanol alone, and freshly separated epidermis was spread on them; the explants mounted on the Differentiation of epidermis in culture 109 collagen gel were then placed on a clot and incubated at 37-5 °C for 2-12 days. All explants were subcultured on to fresh clots every 2 days, and after fixation were examined histologically.

RESULTS 1. Epidermis grown alone, then recombined with dermis Epidermis grown in isolation on a raft developed as described previously (Dodson, 1966). The basal cells very rapidly became flattened, the epithelium thickened, and the cells lost their regular, layered arrangement; some cells showed signs of differentiation, but by 2 days in culture most nuclei were pycnotic (Plate 1, figs. A-D). On Millipore filter the results were variable, some explants developing as above while others became attached to the filter and sometimes formed a layered arrangement. The latter grew slightly differently after recombination and are described separately; the main description refers to epidermis isolated on rafts or unattached to Millipore filter. Thirty-three explants of epidermis were grown in isolation for 24 h, then recombined with dermis. The epidermis was thickened and there were two or three layers of flattened cells at the basal surface. Mitosis had ceased, but the cytoplasm was still basophilic. The periderm, which had migrated round to the lower surface at the edges of some explants, had begun to develop its characteristic granules (Plate 1, fig. A). Within 3 h of the recombination, the epidermis was enveloped by dermal cells, which made close and continuous contact with the basal layer, but not with the periderm. A new periodic acid-Schiff (PAS)- positive and aniline blue-staining basement membrane first appeared under some areas of basal cells at about 10 h after recombination (Plate 2, fig. E), but it was not present under the whole basal layer until 20-27 h. Closely associated with the appearance of the basement membrane were reorientation and division of the basal cells. Although nearly all these elements were flattened at 10 h, after 15 h many were cuboidal and by 20 h most were cuboidal or even columnar (Plate 2, fig. F); flattened cells persisted in some regions, however, even after 4 and 5 days. Mitosis, which had ceased in the isolated epidermis, reappeared in both flattened and cuboidal basal cells at 10-15 h after recombination. The s. basale, re-formed 10-20 h after recombination, pushed up layers of differentiating cells which, together with the layers of flattened cells that developed during isolation, formed a s. spinosum. Meanwhile the upper parts of the epidermis continued to develop as though still isolated and many of the cells became pycnotic, although the lower cells, immediately above the regenerating epithelium, retained their basophilia longer and tended to differentiate further than the more distal cells. Cornified cells first appeared in the regenerating epithelium after 2-3 days (Plate 2, fig. G) and by 5 days a well-arranged keratinizing epithelium had been formed. In the most healthy explants the regenerated epidermis extended over the inner surface of the surrounding dermis, so that a keratinizing pearl was formed, in the centre of which were the remains 110 J. W. DODSON of the upper parts of the isolated epidermis. The dermis, both above and below the epithelium, underwent the normal development found in culture: the cells were healthy and produced more intercellular material; the basement mem- branes remaining on the outer surfaces were sometimes seen, even after 2 days in vitro, but often were not detected. When isolated for 30 h, the histological appearance of the epidermis was similar to that at 24 h, but of thirteen explants isolated for this time, only five redeveloped a viable epithelium when recombined with dermis for 2 days; some cuboidal, dividing basal cells, a few layers of s. spinosum, and a basement membrane were present (Plate 3, fig. H). In the other explants there were occasional small groups of living basal cells on a basement membrane, but there was no continuous s. basale and no stratified arrangement. Except for these basal cells, the rest of the epidermis continued to behave as though still isolated. Although a basement membrane was present after 2 days' recombination, the earliest time of its appearance was not determined. After 36 h of isolation, many epidermal cells, including those that were unoriented between the whorls and also those that were flattened on the lower surface, resembled cells of the lower s. spinosum of normal epidermis; a few of the flattened basal elements were like upper spinous cells but some had pycnotic nuclei (Plate 1, fig. B). The cells of the periderm contained granules and occasional large vacuoles. Twenty pieces of this epidermis were recombined

EXPLANATION OF PLATES The explants are from the scaly, anterior tarso-metatarsal skin of 12-day embryonic chickens; they were grown in organ culture for various periods and were fixed in acetic Zenker's solution. PAS: periodic acid-Schiff technique.

PLATE 1 Fig. A. Epidermis cultivated in isolation for 24 h. The tissue has thickened; the cells in the centre are unoriented and undifferentiated, while those at the lower surface (Fl) are flattened, arranged in layers, and resemble an early s. spinosum. The periderm (P) contains its characteristic granules and in places has migrated round to the basal surface. (Azan: x 310.) Fig. B. Epidermis cultivated in isolation for 36 h. Some cells (S) resemble those of the lower s. spinosum of normal epidermis; others, including some of the flattened lower cells (Fl), have lost their basophilia and may have pycnotic nuclei. P, Periderm. (Celestin blue and Mayer's acid haemalum after PAS: x 310.) Fig. C. Isolated epidermis after 42 h in culture. Most of the basal cells, and also the flattened cells above them (Fl), somewhat resemble those of the upper s. spinosum in that the cytoplasm is pale and the cell outlines are prominent, but often the nuclei are pycnotic. Most of the central cells are still basophilic. (Celestin blue and Mayer's acid haemalum after PAS: x 310.) Fig. D. Isolated epidermis after 48 h in culture. Most cells are degenerate; they are swollen, have empty cytoplasm, and pycnotic nuclei. Some have a little keratinous material at their periphery and only a few cells, arranged in whorls (W), are still basophilic. Fl: Flattened cells on lower surface; P: periderm. (Azan: x 310.) /. Embryol. exp. Morph., Vol. 17, Perl 1 PLATE 1

J. W. DODSON facing p. 110 J. Embryo/, exp. Morph., Vol. 17, Part I PLATE 2

J. W. DODSON facing p. Ill Differentiation of epidermis in culture 111 with dermis and then cultivated for a further 2-7 days. After 2 days, only one piece showed any viable epithelium with a s. spinosum, basal cells, and a basement membrane (Plate 3, fig. I). The other explants contained groups of a few living basal cells, sometimes on a basement membrane, but no organized s. basale (Plate 3, fig. J). In explants cultivated for a longer time, no living epidermal cells were present, indicating that the viable basal cells seen after 2 days of recombination were not able to regenerate a healthy keratinizing epithelium. In epidermis grown alone for 42 h, the changes noted previously had pro- ceeded further. Most of the basal cells had pale cytoplasm and prominent outlines, and often the nuclei were pycnotic (Plate 1, fig. C); these changes were also seen, to a variable extent, in the central region and upper spinous cells. After 2 days' recombination with the dermis, five explants out of seven contained a few living basal cells, including dividing cells, and one had a basement membrane, but in none was there an organized s. basale or stratified arrangement; in the other two explants no living epidermal cells survived. After 48 h in isolation most of the epidermal cells had prominent outlines, empty cytoplasm, and pycnotic nuclei (Plate 1, fig. D). Eight pieces of epidermis were grown alone for this time and then recombined with dermis for 2-7 days. Groups of living basal cells were present in some explants after 2 days, but there were none after 7 days. In eight other explants the isolated epidermis had been attached to Millipore filter and to a varying degree had maintained a stratified structure; it was recombined with dermis after 36 h and after 48 h of isolation. Three days after the recombination the basal cells were alive and occasionally dividing, and they were still living after 5 days, but after 7 days all were dead. No healthy keratinizing epidermis developed. These results show that after 24 h in isolation the epidermis when recombined with dermis is still capable of regenerating a keratinizing epithelium. This ability is even present after 30 h of isolation, although it may be reduced, but

PLATE 2 Fig. E. Epidermis isolated for 24 h, then recombined with living dermis and cultivated for a further 10 h. The dermis (D) is separated from the peridermal surface of the epidermis by a gap (G), but is closely apposed to the lower surface, where a PAS-reactive basement membrane (Bm) has re-formed. The basal cells are still flattened. (PAS after diastase: x490.) Fig. F. Epidermis isolated for 24 hr, then recombined with dermis for a further 20 h. Many of the basal cells (B) have now regained a cuboidal orientation and some are dividing; above them is an early s. spinosum (Ss), but the cells of the upper part of the epithelium are still unoriented, as in epidermis grown alone. D, Dermis; Gl, glycogen. (PAS and Mayer's acid haemalum: x720.) Fig. G. Epidermis isolated for 24 h, then recombined with dermis for a further 2 days. A healthy, keratinizing epithelium has been regenerated, with s. basale (B), s. spinosum (Ss), and s. corneum (5c); above the last are the remains of the upper part of the isolated epidermis, which took no part in the regeneration. D, Dermis. (Azan: x 310.) 112 J. W. DODSON thereafter it is lost. The basal cells may appear viable after being grown in isolation for as long as 48 h, especially if attached to Millipore filter, but they can no longer regenerate a healthy epidermis. The relationship between the orientation of the basal cells while isolated and the subsequent differentiation of the epidermis is summarized in Table 1.

2. The effect of excess of vitamin A upon epidermis grown on a collagen gel Epidermis growing on collagen gels in the presence of excess of vitamin A (twenty explants) spread rapidly over the gel and also into the surrounding clot. The clot underlying the epithelium was lysed, but there was no detectable free mucus associated with vitamin-treated explants. On control medium, epidermis spread over the gel less rapidly and did not extend over the clot, which was not lysed. After 3-4 days in vitro the control explants showed the opacity that indicates cornification (Fell, 1957). Histological examination showed that control cultures keratinized in the same way as on normal medium (Plate 4, fig. K). This process was completely inhibited by both doses of the vitamin, although the centres of the treated explants contained layers of flattened cells, as did epidermis with living dermis in the presence of the vitamin. The superficial cells were very greatly swollen and above them were the remains of the periderm. After 10-12 days in culture, as a result of the extension of the epidermis, most of the treated epithelium was extremely flattened and only one or two cells thick. Some of the flattened cells contained PAS-positive mucous material (Plate 4, fig. M) at least some of which stained with alcian blue, indicating that it was acidic. A few cells contained refractile droplets which stained with the PAS technique and with azocarmine; these appeared to be related to the peculiar secretion that is sometimes produced by the periderm (McLoughlin, 1961Z?; Fell, 1962). Mitoses were more common than in control cultures and there were occasional pigment cells. After treatment at the higher dose of the vitamin the effects were more marked: the epidermis spread further and so was thinner, each cell extending over a large

PLATE 3 Fig. H. Epidermis grown alone for 30 h, then recombined with living dermis and cultivated for a further 2 days. A stratified epithelium has been redeveloped in the lower part of the epidermis, but the upper part has degenerated as though still isolated. B, Basal cells; D, dermis; Ss, s. spinosum. (Haematoxylin and eosin: x 310.) Fig. I. Epidermis isolated for 36 h, then recombined with living dermis and cultivated for a further 2 days. In this explant there is a stratified epithelium with dividing basal cells (B) and a s. spinosum (Ss), although most of the epidermis has degenerated as if still isolated. D, Dermis; M, mitosis. (Haematoxylin and eosin: x 310.) Fig. J. Epidermis treated as in Fig. I. Here the few living cells are not organized into layers. D, Dermis. (Mayer's acid haemalum after PAS: x 310.) J. Embryo/, exp. Morph., Vol. 17, Part 1 PLATE 3

J. W. DODSON facing p. 112 J. Embryo!, exp. Morph., Vol. 17, Part 1 PLATE 4

J. W. DODSON facing p. 113 Differentiation of epidermis in culture 113 area (Plate 4, fig. N) and pigment cells were common. These features were not observed in the cultures grown on control medium. The behaviour and differentiation of the epidermis grown on collagen gels in the presence of excess of vitamin A was essentially similar to that of epidermis growing with living dermis under the same conditions (Plate 4, fig. L); the only significant difference was the greater spreading on collagen gels.

DISCUSSION The present results extend previous observations in showing that the epidermis of the embryonic chicken foot is a flexible system whose differentiation can be altered. Despite the flattening and signs of differentiation that occur in the lower cells when the epidermis is grown alone for 24-30 h, the basal cells can still regenerate a healthy keratinizing epithelium if recombined with dermis. Beyond that time, the regenerative ability is lost, but even after 42 h cultivation in isolation, on recombination the basal cells are able to become cuboidal and divide, and a basement membrane may be formed. After this period, these properties also disappear, although a few cells retain their basophilia for a short while. Thus for a time the changes that occur on isolation are reversible, but there is then a gradual loss of the potentialities of the lowermost cells. It should be noted that regeneration is effected mainly by the basal cells. The more superficial elements fail to recover when the epidermis is recombined, even after only 24 h isolation, and degenerate as though still segregated. It is interesting that mitosis, which ceases in basal cells almost immediately after isolation and in the central region about 16 h later, is resumed in the basal layer on recombination after as long as 42 h in isolation alone. This confirms and extends Wessells' observation (1963) that dividing basal cells appeared on recombination with dermis after 24 h in isolation. It would seem that the shape of the basal cells is not necessarily related to the

PLATE 4 Fig. K. Epidermis on collagen gel, cultivated on control medium for 9 days. The epidermis has many layers of cells and has formed a s. corneum (Sc). Cg, Collagen gel. (Azan: x 1180.) Fig. L. Epidermis on living dermis in the presence of excess of vitamin A (2-4 i.u./ml); 10 days in culture. The epithelium is only a few cells thick and the cells are very attenuated. Keratinization has been inhibited and mucous droplets are present. D, Dermis; E, epidermis; Mu, PAS-reactive mucus. (PAS after diastase, followed by Mayer's acid haemalum: x 740.) Fig. M. Epidermis on collagen gel in the presence of excess of vitamin A (2-4 i.u./ml); 12 days in vitro. The thin epithelium resembles the control on living dermis (Fig. L). Keratini- zation has been inhibited and there are droplets of PAS-reactive mucus (Mu) (cf. fig. K). Cg, Collagen gel. (PAS after diastase, followed by Mayer's acid haemalum: x 750.) Fig. N. Epidermis on collagen gel in the presence of excess of vitamin A (9 i.u./ml); 9 days in culture. The epidermis has spread greatly so that most of it is only one cell thick. Some cells contain PAS-reactive mucous droplets (Mu) (cf. figs. K, M). Cg, Collagen gel. (PAS and Mayer's acid haemalum: xll90.) 8 JEEM 1J 114 J. W. DODSON differentiation of the epithelium, for under experimental manipulation various associations of orientation and differentiation occur (Table 1). The development of the stratified organization seems to depend, rather, on the polarization of the epithelium. In isolation the epidermis becomes bipolar, grading from a central undifferentiated region to partially differentiated cells on either surface; thus, for the basal cells, the direction of polarity of differentiation is opposite to the normal. On recombination of epidermis grown alone for 24 h this reversal can be changed back to the usual situation, but the finding that, after 30-36 h in isolation, the lowermost cells can return to normal polarity yet fail to regenerate a healthy epithelium indicates that normal polarity alone is not enough for typical epidermal differentiation.

Table 1. Relationships between orientation of the basal cells and differentiation of the epidermis in embryonic chicken skin

Orientation of Development of Experimental conditions basal cells epidermis 1 Normal skin Columnar Differentiating 2 Epidermis on dermis in vitro (e.g. Cuboidal Differentiating Fell, 1957; Dodson, 1966) 3 Epidermis on collagen gel (Dodson, Flattened Differentiating 1966) 4 Epidermis in isolation (Wessells, Flattened Non-differentiating 1962; Dodson, 1966) 5 Epidermis isolated for 24 h, then Flattened, then becoming Differentiating recombined with dermis cuboidal 6 Epidermis isolated for 36 h, then Flattened, then becoming Non-differentiating recombined with dermis cuboidal 7 Epidermis isolated for 48 h, then Flattened Non-differentiating recombined with dermis

The appearance of a new basement membrane was one of the first events following reassociation of epidermis and dermis, but in the present experiments it formed more slowly against the isolated tissue than against the freshly separated epithelium (Dodson, 1966). This delay may be related to the reversed polarity of the basal cells. For the survival and subsequent development of the basal cells of this metatarsal epidermis, it is clearly not necessary for the basement membrane to be present continuously. It can be absent for 24-30 hours; after that period, however, the basal cells lose their potentiality for regeneration, even though a new basement membrane is still re-formed against them. Epidermis grown on collagen gels in the presence of excess of vitamin A resembled that on living dermis in such conditions, for in both cases keratinization was inhibited, the epidermis spread out (although to a greater degree on the Differentiation of epidermis in culture 115 gel), secretory cells formed, mitosis was more frequent than in control explants, and pigment cells developed. The appearance of pigment in chicken epidermis in culture is a common concomitant of treatment with vitamin A (Fell, 1956,1962); its presence in epidermis grown on a collagen gel shows that the production of pigment by epithelium of this age does not require the presence of dermal cells. It is interesting that the cells of the vitamin-treated epidermis were able to leave the gel and migrate within the plasma clot, a phenomenon not seen in control explants when placed either on a gel or directly on the clot. This indicates that in the presence of the vitamin the requirements of the basal cells for a sub- stratum are much less specific. The increased spreading of treated epidermis may be related to a lack of cohesion between cells, for in the epidermis of whole skin treated with vitamin A there are fewer desmosomes than in control explants (Fitton Jackson & Fell, 1963). Since on collagen gels the vitamin-treated epidermis differed considerably from that in control medium, it is clear that the vitamin can affect the epidermis directly, without the intervention of dermal cells. This result, obtained with older epidermis on a collagen gel, extends the observations of McLoughlin (1961 a) on the isolated epidermis of 5-day chicken embryos explanted directly on a plasma-embryo extract clot. The keratinization of older epidermis on collagen gels on normal medium and its secretory transformation in the presence of excess of vitamin A demonstrate that the capacity for differentiation, whether normal or otherwise, resides in the epidermal cells.

SUMMARY 1. Two series of experiments were made to determine the possible alteration of paths of differentiation of the epidermis from the metatarsal region of 12-day chicken embryos. The epidermis, separated from dermis after Versene treatment of the skin, either was cultivated in isolation for various periods, then recombined with dermis and cultivated further (series 1), or was placed on gels of collagen and grown in culture in the presence of excess of vitamin A (series 2). All explants were examined histologically. 2. When epidermis isolated for 24 h was recombined with dermis, the upper cells degenerated as though still in isolation, but the basal cells, although they had become flattened while isolated, regenerated a viable, keratinizing epidermis; this ability was lost after 30-36 h of isolation. A basement membrane was re-formed against epidermis, following recombination, after isolation for 36-42 h. Thus the basal cells of isolated epidermis can survive for several hours in the absence of a basement membrane; the changes that they undergo are reversible for 24-30 h, but there is then a gradual loss of their potentialities. 3. Epidermis grown on collagen gels on control medium formed a stratum corneum, but in the presence of excess of vitamin A no keratin appeared, the epithelium became thin, and mucous cells developed. The effect resembled that 8-2 116 J. W. DODSON on epidermis of whole skin. The results demonstrate that the effect of the vitamin on the epidermis is direct and is not mediated by the dermis.

RESUME La differenciation de Vepiderme. II. Modes alternatifs de differenciation de Vepiderme embryonnaire de poulet en culture d'organes 1. On a realise deux series d'experiences pour determiner la possibility de modifier la differenciation de l'epiderme de region metatarsienne d'embryons de poulet de 12 jours. L'epiderme, separe du derme apres un traitement de la peau au Versene, ou bien a ete cultive isolement pendant diverses durees, puis recombine avec du derme et de nouveau cultive (serie 1), ou bien a ete place sur des gels de collagene et cultive en presence d'un exces de vitamine A (serie 2). Tous les explants ont ete examines histologiquement. 2. Quand de l'epiderme isole pendant 24 heures a ete recombine avec du derme, les cellules superieures ont degenere comme si elles etaient encore isolees, mais les cellules basales, bien qu'elles se fussent aplaties lorsqu'elles etaient isolees, ont regenere un epiderme viable, formant de la keratine; cette aptitude a ete perdue apres 30 a 36 heures d'isolement. Une membrane basale s'est reformee contre l'epiderme, a la suite de la recombinaison, apres un isolement de 36 a 42 heures. Ainsi les cellules basales de l'epiderme isole peuvent survivre pendant plusieurs heures en l'absence d'une membrane basale; les modifications qu'elles subissent sont reversibles pendant 24 a 30 heures, mais une perte graduelle de leurs potentialites survient ensuite. 3. L'epiderme cultive sur gels de collagene et sur milieu temoin a forme un stratum corneum, mais en presence d'un exces de vitamine A il n'est pas apparu de keratine, l'epiderme est devenu mince et des cellules muqueuses se sont developpees. L'effet ressemblait a celui qu'on obtient sur l'epiderme de peau complete. Les resultats demontrent que 1'action de la vitamine A sur l'epiderme est direct et n'est pas transmise par l'intermediaire du derme.

I thank the Medical Research Council for a research scholarship and I am deeply indebted to Professor Dame Honor Fell, D.B.E., F.R.S., to Dr S. Fitton Jackson, and to Dr A. Gliicksmann for their very helpful discussions and criticism.

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(Manuscript received 14 July 1966)