/. Embryol. exp. Morph., Vol. 17, 1, pp. 107-117, February 1967
With 4 plates
Printed in Great Britain
107
The differentiation of epidermis
II. Alternative pathways of differentiation of embryonic
chicken epidermis in organ culture
ByJ. W. DODSON 1
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 cellsfirstappeared 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 membranes 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
J. W. DODSON
PLATE 1
facing p. 110
J. Embryo/, exp. Morph., Vol. 17, Part I
J. W. DODSON
PLATE 2
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 proceeded 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
J. W. DODSON
PLATE 3
facing p. 112
J. Embryo!, exp. Morph., Vol. 17, Part 1
J. W . D O D S O N
PLATE 4
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). Keratinization 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
Experimental conditions
1 Normal skin
2 Epidermis on dermis in vitro (e.g.
Fell, 1957; Dodson, 1966)
3 Epidermis on collagen gel (Dodson,
1966)
4 Epidermis in isolation (Wessells,
1962; Dodson, 1966)
5 Epidermis isolated for 24 h, then
recombined with dermis
6 Epidermis isolated for 36 h, then
recombined with dermis
7 Epidermis isolated for 48 h, then
recombined with dermis
Orientation of
basal cells
Development of
epidermis
Columnar
Cuboidal
Differentiating
Differentiating
Flattened
Differentiating
Flattened
Non-differentiating
Flattened, then becoming Differentiating
cuboidal
Flattened, then becoming Non-differentiating
cuboidal
Flattened
Non-differentiating
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 substratum 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.
REFERENCES
J. W. (1963). On the nature of tissue interactions in embryonic skin. Expl Cell Res.
31, 233-5.
DODSON, J. W. (1967). The differentiation of epidermis. I. The interrelationship of epidermis
and 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 skin
explanted at different stages of differentiation. Proc. R. Soc. B, 146, 242-56.
DODSON,
Differentiation of epidermis in culture
117
FELL, H. B. (1962). The influence of hydrocortisone on the metaplastic action of vitamin A
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 by
high vitamin A. /. Physiol. 119, 470-88.
FELL, H. B. & ROBISON, R. (1929). The growth, development, and phosphatase activity of
embryonic avian femora and limb buds cultivated in vitro. Biochem. J. 23, 767-84.
FITTON JACKSON, S. & FELL, H. B. (1963). Epidermalfinestructure in embryonic chicken skin
during 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 of
the chick embryo. /. Morph. 88, 49-92.
MCLOUGHLIN, C. B. (1961 a). The importance of mesenchymal factors in the differentiation
of chick epidermis. I. The differentiation in culture of the isolated epidermis of the embryonic
chick and its response to vitamin A. /. Embryol. exp. Morph. 9, 370-84.
MCLOUGHLIN, C. B. (19616). The importance of mesenchymal factors in the differentiation
of chick epidermis. II. Modifications of epidermal differentiation by contact with different
types of mesenchyme. J. Embryol. exp. Morph. 9, 385-409.
SCHAFFER, B. M. (1956). The culture of organs from the embryonic chick on cellulose-acetate
fabric. 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 thymidine
by embryonic epidermis. Expl Cell Res. 30, 36-55.
(Manuscript received 14 July 1966)