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

Printed in Great Britain

The differentiation of epidermis

II. Alternative pathways of differentiation of embryonicchicken 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 investigatethe role of the dermis in determining alternative pathways of differentiation inthe epidermis. Previous work has shown that the scaly metatarsal epidermis of12-day chicken embryos, when isolated in culture on a plasma clot or variousother substrata, fails to develop normally and undergoes a characteristicsequence 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 questionsarise of whether the changes produced in the epidermis by growth in isolationcan be reversed by subsequent recombination of the epithelium with the dermis,and if so at what stage the degeneration becomes irreversible. Accordingly, inthe first series of experiments, epidermis was cultivated in isolation for variousperiods of time, then recombined with dermis and cultivated further; theexplants were examined histologically for signs of a keratinizing epithelium. Ina similar experiment, Wessells (1963) found that proliferating columnar basalcells appeared in epidermis isolated for 10 or 24 h, then recombined with dermisfor a further 1 or 2 days, but longer periods of segregation were not tested, norwas further differentiation described.

The differentiation of the epidermis of embryonic chicken skin in culture canalso 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 embryoswas treated, keratinization was inhibited and a mucous metaplasia was in-duced; the latter change, however, was less extensive and less frequent in theoldest skin. Originally it was not known whether this action of the vitamin wasdirectly on the epidermis or whether it was mediated through changes producedin 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 withslight keratinization in control explants were inhibited by the vitamin and in thetreated explants a thin layer of mucus appeared; this result indicated that in thisvery young material the changes were due to a primary action of the vitamin onthe epithelium. The second series of experiments recorded in the present paperwas made to determine whether the vitamin also acts directly on the partlydifferentiated epidermis of older embryos and whether the isolated epitheliumfrom such embryos can undergo a full mucous metaplasia when grown on acollagen gel in the presence of excess of vitamin A.

MATERIALS AND METHODS

Epidermis was separated from dermis after Versene treatment of the scalyskin of the anterior tarso-metatarsal region of 12-day chicken embryos (stages37^-38^ of Hamburger & Hamilton, 1951). The techniques of separation,cultivation, and histological examination have been described in a previouspaper (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 thewatch-glass technique of Fell & Robison (1929) on clots made of 12 drops offowl plasma and 8 drops of embryo extract. The pieces of separated dermiswere placed on rayon rafts on clots and kept at room temperature (18-20 °C)until required for recombination, except that when epidermis was isolated for48 h, dermis was prepared freshly immediately before recombination.

After incubation the epidermis was carefully removed from the raft or filterand was placed on the inner surface of a piece of dermis (i.e. the surface notbearing the basement membrane) on a raft; a second piece of dermis was thenplaced over the epithelium, again with its inner surface next to the latter tissue.This 'sandwich' technique obviated the necessity of determining the basalsurface of the twisted sheet of isolated epidermis, and placing the inner surfaceof the dermis next to the epithelium prevented the extant basement membraneon the dermis from being confused with any newly formed basement membranenext 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 aceticacid-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 togive a concentration of either 2-4 or 9 i.u. per ml in the final plasma-embryoextract clot. The medium for control explants contained the same amount ofethanol (0-1 %). Pieces of collagen gel were soaked in clot exudate (H. B. Fell,personal communication) containing either vitamin A or ethanol alone, andfreshly 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 fixationwere 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 epitheliumthickened, and the cells lost their regular, layered arrangement; some cellsshowed signs of differentiation, but by 2 days in culture most nuclei werepycnotic (Plate 1, figs. A-D). On Millipore filter the results were variable, someexplants developing as above while others became attached to the filter andsometimes formed a layered arrangement. The latter grew slightly differentlyafter recombination and are described separately; the main description refersto epidermis isolated on rafts or unattached to Millipore filter.

Thirty-three explants of epidermis were grown in isolation for 24 h, thenrecombined with dermis. The epidermis was thickened and there were two orthree layers of flattened cells at the basal surface. Mitosis had ceased, but thecytoplasm was still basophilic. The periderm, which had migrated round to thelower surface at the edges of some explants, had begun to develop its charac-teristic granules (Plate 1, fig. A). Within 3 h of the recombination, the epidermiswas enveloped by dermal cells, which made close and continuous contact withthe basal layer, but not with the periderm. A new periodic acid-Schiff (PAS)-positive and aniline blue-staining basement membrane first appeared undersome areas of basal cells at about 10 h after recombination (Plate 2, fig. E), butit was not present under the whole basal layer until 20-27 h. Closely associatedwith the appearance of the basement membrane were reorientation and divisionof 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 after4 and 5 days. Mitosis, which had ceased in the isolated epidermis, reappeared inboth flattened and cuboidal basal cells at 10-15 h after recombination.

The s. basale, re-formed 10-20 h after recombination, pushed up layers ofdifferentiating cells which, together with the layers of flattened cells that de-veloped during isolation, formed a s. spinosum. Meanwhile the upper parts ofthe epidermis continued to develop as though still isolated and many of thecells became pycnotic, although the lower cells, immediately above the regene-rating epithelium, retained their basophilia longer and tended to differentiatefurther than the more distal cells. Cornified cells first appeared in the regeneratingepithelium after 2-3 days (Plate 2, fig. G) and by 5 days a well-arranged kera-tinizing epithelium had been formed. In the most healthy explants the re-generated 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 belowthe epithelium, underwent the normal development found in culture: the cellswere healthy and produced more intercellular material; the basement mem-branes remaining on the outer surfaces were sometimes seen, even after 2 daysin vitro, but often were not detected.

When isolated for 30 h, the histological appearance of the epidermis wassimilar to that at 24 h, but of thirteen explants isolated for this time, only fiveredeveloped a viable epithelium when recombined with dermis for 2 days; somecuboidal, dividing basal cells, a few layers of s. spinosum, and a basementmembrane were present (Plate 3, fig. H). In the other explants there wereoccasional small groups of living basal cells on a basement membrane, but therewas no continuous s. basale and no stratified arrangement. Except for thesebasal cells, the rest of the epidermis continued to behave as though still isolated.Although a basement membrane was present after 2 days' recombination, theearliest time of its appearance was not determined.

After 36 h of isolation, many epidermal cells, including those that wereunoriented between the whorls and also those that were flattened on the lowersurface, resembled cells of the lower s. spinosum of normal epidermis; a fewof the flattened basal elements were like upper spinous cells but some hadpycnotic nuclei (Plate 1, fig. B). The cells of the periderm contained granules andoccasional 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 embryonicchickens; they were grown in organ culture for various periods and were fixed in aceticZenker'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 thecentre 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 charac-teristic 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 lowers. 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 andMayer's acid haemalum after PAS: x 310.)Fig. C. Isolated epidermis after 42 h in culture. Most of the basal cells, and also the flattenedcells above them (Fl), somewhat resemble those of the upper s. spinosum in that the cyto-plasm is pale and the cell outlines are prominent, but often the nuclei are pycnotic. Most ofthe 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 theirperiphery and only a few cells, arranged in whorls (W), are still basophilic. Fl: Flattened cellson 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 onepiece showed any viable epithelium with a s. spinosum, basal cells, and a base-ment membrane (Plate 3, fig. I). The other explants contained groups of afew living basal cells, sometimes on a basement membrane, but no organizeds. basale (Plate 3, fig. J). In explants cultivated for a longer time, no living epi-dermal cells were present, indicating that the viable basal cells seen after 2 daysof 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 out-lines, and often the nuclei were pycnotic (Plate 1, fig. C); these changes were alsoseen, to a variable extent, in the central region and upper spinous cells. After2 days' recombination with the dermis, five explants out of seven contained afew living basal cells, including dividing cells, and one had a basement mem-brane, but in none was there an organized s. basale or stratified arrangement; inthe 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 epidermiswere 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 therewere none after 7 days.

In eight other explants the isolated epidermis had been attached to Milliporefilter and to a varying degree had maintained a stratified structure; it was recom-bined with dermis after 36 h and after 48 h of isolation. Three days after therecombination the basal cells were alive and occasionally dividing, and theywere still living after 5 days, but after 7 days all were dead. No healthy keratini-zing epidermis developed.

These results show that after 24 h in isolation the epidermis when recombinedwith dermis is still capable of regenerating a keratinizing epithelium. Thisability 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 afurther 10 h. The dermis (D) is separated from the peridermal surface of the epidermis by agap (G), but is closely apposed to the lower surface, where a PAS-reactive basement mem-brane (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. Manyof the basal cells (B) have now regained a cuboidal orientation and some are dividing; abovethem is an early s. spinosum (Ss), but the cells of the upper part of the epithelium are stillunoriented, as in epidermis grown alone. D, Dermis; Gl, glycogen. (PAS and Mayer's acidhaemalum: x720.)Fig. G. Epidermis isolated for 24 h, then recombined with dermis for a further 2 days. Ahealthy, 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 inisolation for as long as 48 h, especially if attached to Millipore filter, but theycan no longer regenerate a healthy epidermis. The relationship between theorientation of the basal cells while isolated and the subsequent differentiation ofthe epidermis is summarized in Table 1.

2. The effect of excess of vitamin A upon epidermis grown ona 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 freemucus associated with vitamin-treated explants. On control medium, epidermisspread over the gel less rapidly and did not extend over the clot, which was notlysed. After 3-4 days in vitro the control explants showed the opacity that indi-cates cornification (Fell, 1957).

Histological examination showed that control cultures keratinized in thesame way as on normal medium (Plate 4, fig. K). This process was completelyinhibited by both doses of the vitamin, although the centres of the treatedexplants contained layers of flattened cells, as did epidermis with living dermisin the presence of the vitamin. The superficial cells were very greatly swollenand 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 wasextremely flattened and only one or two cells thick. Some of the flattened cellscontained PAS-positive mucous material (Plate 4, fig. M) at least some of whichstained with alcian blue, indicating that it was acidic. A few cells containedrefractile droplets which stained with the PAS technique and with azocarmine;these appeared to be related to the peculiar secretion that is sometimes producedby the periderm (McLoughlin, 1961Z?; Fell, 1962). Mitoses were more commonthan in control cultures and there were occasional pigment cells. After treat-ment at the higher dose of the vitamin the effects were more marked: theepidermis 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 cultivatedfor a further 2 days. A stratified epithelium has been redeveloped in the lower part of theepidermis, 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 afurther 2 days. In this explant there is a stratified epithelium with dividing basal cells (B) anda 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 P L A T E 4

J. W . D O D S O N facing p. 113

Differentiation of epidermis in culture 113area (Plate 4, fig. N) and pigment cells were common. These features were notobserved in the cultures grown on control medium. The behaviour and differ-entiation of the epidermis grown on collagen gels in the presence of excess ofvitamin A was essentially similar to that of epidermis growing with livingdermis under the same conditions (Plate 4, fig. L); the only significant differencewas the greater spreading on collagen gels.

DISCUSSION

The present results extend previous observations in showing that the epidermisof the embryonic chicken foot is a flexible system whose differentiation can bealtered. Despite the flattening and signs of differentiation that occur in the lowercells when the epidermis is grown alone for 24-30 h, the basal cells can stillregenerate a healthy keratinizing epithelium if recombined with dermis. Beyondthat time, the regenerative ability is lost, but even after 42 h cultivation in isola-tion, on recombination the basal cells are able to become cuboidal and divide,and a basement membrane may be formed. After this period, these propertiesalso 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 isthen a gradual loss of the potentialities of the lowermost cells. It should be notedthat regeneration is effected mainly by the basal cells. The more superficialelements fail to recover when the epidermis is recombined, even after only 24 hisolation, and degenerate as though still segregated.

It is interesting that mitosis, which ceases in basal cells almost immediatelyafter isolation and in the central region about 16 h later, is resumed in the basallayer on recombination after as long as 42 h in isolation alone. This confirmsand extends Wessells' observation (1963) that dividing basal cells appeared onrecombination 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 epidermishas 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 daysin culture. The epidermis has spread greatly so that most of it is only one cell thick. Some cellscontain PAS-reactive mucous droplets (Mu) (cf. figs. K, M). Cg, Collagen gel. (PAS andMayer's acid haemalum: xll90.)

8 JEEM 1J

114 J. W. DODSON

differentiation of the epithelium, for under experimental manipulation variousassociations of orientation and differentiation occur (Table 1). The develop-ment of the stratified organization seems to depend, rather, on the polarizationof the epithelium. In isolation the epidermis becomes bipolar, grading from acentral undifferentiated region to partially differentiated cells on either surface;thus, for the basal cells, the direction of polarity of differentiation is oppositeto the normal. On recombination of epidermis grown alone for 24 h thisreversal can be changed back to the usual situation, but the finding that, after30-36 h in isolation, the lowermost cells can return to normal polarity yet failto regenerate a healthy epithelium indicates that normal polarity alone is notenough for typical epidermal differentiation.

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

12

3

4

5

6

7

Experimental conditions

Normal skinEpidermis on dermis in vitro (e.g.Fell, 1957; Dodson, 1966)

Epidermis on collagen gel (Dodson,1966)

Epidermis in isolation (Wessells,1962; Dodson, 1966)

Epidermis isolated for 24 h, thenrecombined with dermis

Epidermis isolated for 36 h, thenrecombined with dermis

Epidermis isolated for 48 h, thenrecombined with dermis

Orientation ofbasal cells

ColumnarCuboidal

Flattened

Flattened

Flattened, then becomingcuboidal

Flattened, then becomingcuboidal

Flattened

Development ofepidermis

DifferentiatingDifferentiating

Differentiating

Non-differentiating

Differentiating

Non-differentiating

Non-differentiating

The appearance of a new basement membrane was one of the first eventsfollowing reassociation of epidermis and dermis, but in the present experimentsit formed more slowly against the isolated tissue than against the freshlyseparated epithelium (Dodson, 1966). This delay may be related to the reversedpolarity of the basal cells. For the survival and subsequent development of thebasal cells of this metatarsal epidermis, it is clearly not necessary for the base-ment 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 Aresembled that on living dermis in such conditions, for in both cases keratiniza-tion 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 inculture 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 ofpigment 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 leavethe gel and migrate within the plasma clot, a phenomenon not seen in controlexplants when placed either on a gel or directly on the clot. This indicates thatin 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 maybe related to a lack of cohesion between cells, for in the epidermis of whole skintreated with vitamin A there are fewer desmosomes than in control explants(Fitton Jackson & Fell, 1963).

Since on collagen gels the vitamin-treated epidermis differed considerablyfrom that in control medium, it is clear that the vitamin can affect the epidermisdirectly, without the intervention of dermal cells. This result, obtained with olderepidermis on a collagen gel, extends the observations of McLoughlin (1961 a)on the isolated epidermis of 5-day chicken embryos explanted directly on aplasma-embryo extract clot. The keratinization of older epidermis on collagengels on normal medium and its secretory transformation in the presence ofexcess of vitamin A demonstrate that the capacity for differentiation, whethernormal or otherwise, resides in the epidermal cells.

SUMMARY

1. Two series of experiments were made to determine the possible alterationof paths of differentiation of the epidermis from the metatarsal region of 12-daychicken embryos. The epidermis, separated from dermis after Versene treatmentof the skin, either was cultivated in isolation for various periods, then recombinedwith dermis and cultivated further (series 1), or was placed on gels of collagenand grown in culture in the presence of excess of vitamin A (series 2). All explantswere examined histologically.

2. When epidermis isolated for 24 h was recombined with dermis, the uppercells degenerated as though still in isolation, but the basal cells, although theyhad become flattened while isolated, regenerated a viable, keratinizing epi-dermis; this ability was lost after 30-36 h of isolation. A basement membranewas re-formed against epidermis, following recombination, after isolation for36-42 h. Thus the basal cells of isolated epidermis can survive for several hoursin the absence of a basement membrane; the changes that they undergo arereversible 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 stratumcorneum, but in the presence of excess of vitamin A no keratin appeared, theepithelium 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 thevitamin on the epidermis is direct and is not mediated by the dermis.

RESUME

La differenciation de Vepiderme. II. Modes alternatifs de differenciationde Vepiderme embryonnaire de poulet en culture d'organes

1. On a realise deux series d'experiences pour determiner la possibility demodifier la differenciation de l'epiderme de region metatarsienne d'embryonsde poulet de 12 jours. L'epiderme, separe du derme apres un traitement de lapeau au Versene, ou bien a ete cultive isolement pendant diverses durees, puisrecombine avec du derme et de nouveau cultive (serie 1), ou bien a ete place surdes 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 duderme, les cellules superieures ont degenere comme si elles etaient encoreisolees, mais les cellules basales, bien qu'elles se fussent aplaties lorsqu'ellesetaient isolees, ont regenere un epiderme viable, formant de la keratine; cetteaptitude a ete perdue apres 30 a 36 heures d'isolement. Une membrane basales'est reformee contre l'epiderme, a la suite de la recombinaison, apres un isole-ment de 36 a 42 heures. Ainsi les cellules basales de l'epiderme isole peuventsurvivre pendant plusieurs heures en l'absence d'une membrane basale; lesmodifications qu'elles subissent sont reversibles pendant 24 a 30 heures, maisune perte graduelle de leurs potentialites survient ensuite.

3. L'epiderme cultive sur gels de collagene et sur milieu temoin a forme unstratum corneum, mais en presence d'un exces de vitamine A il n'est pas apparude keratine, l'epiderme est devenu mince et des cellules muqueuses se sontdeveloppees. L'effet ressemblait a celui qu'on obtient sur l'epiderme de peaucomplete. Les resultats demontrent que 1'action de la vitamine A sur l'epidermeest 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 indebtedto 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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DODSON, J. W. (1967). The differentiation of epidermis. I. The interrelationship of epidermisand dermis in embryonic chicken skin. / . Embryol. exp. Morph. 17, 83-105.

FELL, H. B. (1956). Effect of excess vitamin A on organized tissues cultivated in vitro. Br.med. Bull. 12, 35-7.

FELL, H. B. (1957). The effect of excess vitamin A on cultures of embryonic chicken skinexplanted at different stages of differentiation. Proc. R. Soc. B, 146, 242-56.

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on the epidermis of embryonic chicken skin in organ culture. / . Embryol. exp. Morph.10, 389^09.

FELL, H. B. & MELLANBY, E. (1953). Metaplasia produced in cultures of chick ectoderm byhigh vitamin A. / . Physiol. 119, 470-88.

FELL, H. B. & ROBISON, R. (1929). The growth, development, and phosphatase activity ofembryonic avian femora and limb buds cultivated in vitro. Biochem. J. 23, 767-84.

FITTON JACKSON, S. & FELL, H. B. (1963). Epidermal fine structure in embryonic chicken skinduring atypical differentiation induced by vitamin A in culture. Devi Biol. 7, 394-419.

HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development ofthe chick embryo. / . Morph. 88, 49-92.

MCLOUGHLIN, C. B. (1961 a). The importance of mesenchymal factors in the differentiationof chick epidermis. I. The differentiation in culture of the isolated epidermis of the embryonicchick and its response to vitamin A. / . Embryol. exp. Morph. 9, 370-84.

MCLOUGHLIN, C. B. (19616). The importance of mesenchymal factors in the differentiationof chick epidermis. II. Modifications of epidermal differentiation by contact with differenttypes of mesenchyme. J. Embryol. exp. Morph. 9, 385-409.

SCHAFFER, B. M. (1956). The culture of organs from the embryonic chick on cellulose-acetatefabric. Expl Cell Res. 11, 244-8.

WESSELLS, N. K. (1962). Tissue interactions during skin histodifferentiation. Devi Biol. 4,87-107.

WESSELLS, N. K. (1963). Effects of extra-epithelial factors on the incorporation of thymidineby embryonic epidermis. Expl Cell Res. 30, 36-55.

(Manuscript received 14 July 1966)