/. Embryol. exp. Morph. Vol. 19, 2, pp. 157-80, April 1968
With 4 plates
\ 57
Printed in Great Britain
Glandular metaplasia
of hair follicles and other responses to vitamin A
excess in cultures of rodent skin
By MARGARET H. HARDY1
From the Department of Anatomy, College of Physicians and Surgeons,
Columbia University, New York
INTRODUCTION
The demonstration of mucous metaplasia in chicken embryonic epidermis
exposed to an excess of vitamin A (Fell & Mellanby, 1953) stimulated many
further investigations (reviewed by Fell, 1964; Fell & Rinaldini, 1965; Dingle &
Lucy, 1965), leading to new insights into control of differentiation and function
at the cellular level.
The object of the present experiments was to study the effects of excess vitamin
A on another keratin-producing system, the developing hair follicle, in organotypic cultures. Hairs and their follicles have received little attention from other
investigators of vitamin A effects in vitro, although Fell & Mellanby (1953)
referred to ' evidence of an inhibitory effect on hair formation' in some preliminary experiments with mouse embryonic skin, and New (1963) reported
that 'usually the development of hair follicles was suppressed' in cultures of
embryonic skin from the rat and mouse, both in the presence and absence of
excess vitamin A.
Some unexpected and dramatic metaplastic changes in hair follicles exposed
to an excess of vitamin A are described in this paper. New information obtained
in the course of the same experiments about the effects of vitamin A on mammalian epidermis (Hardy, 1965, 1967 a) and tissues of the oral cavity (Hardy,
1967 c) are reported elsewhere.
MATERIALS AND METHODS
The mouse embryos used and the four experiments performed have already
been described in the paper dealing with the epidermis (Hardy, 1967 a). The
experiments are summarized in Table 1. Briefly, organotypic cultures of abdominal skin, upper lip and lower jaw were grown in a solid medium consisting of
1
Author's address: Department of Anatomy, University of Guelph, Guelph, Ontario,
Canada.
II
JEEM ig
158
M. H. HARDY
3 drops of fowl plasma and 1 drop of chicken embryo extract on coverslips
inverted over Maximow slides and incubated at 35-5 °C. In each experiment the
effect of adding certain concentrations of vitamin A was tested, and in Exp. 4,
hydrocortisone was added to two of the four treatment groups to see whether
vitamin A effects were suppressed. The explants were transferred to fresh clots
every 3 or 4 days and subdivided when necessary to meet nutritional requirements.
Table 1. Summary of experiments and numbers of explants
Number of explants with additions to basic medium
Age of DuraExp. embryos tion
no. (days) (days)
1
2
15
13|
11
11
Tissue
explanted
Trunk skin
Trunk skin
Upper lip
Half lower
jaw
3
14
14 Upper lip
Half lower
jaw
4
15
11 Half upper
lip
Total explants for each treatment
None Eth
8
Eth
Eth Eth Eth 12-5A Eth Eth
HC 6-2A* 12-5A HC 25-OA 37-5A
8
—
—
1
1
—
—
—
—
8 —
8
4
—
4
1
—
—
1
—
—
—
—
4
4
—
—
—
—
4
4
—
6
6
8
32
—
—
8
—
—
4
—
6
6
4
_
—
6
28
6
—
4
—
—
_
—
_
—
—
—
12
4
Eth = ethanol (00015 ml./ml. final medium); A = vitamin A; HC = hydrocortisone
(7-5 /tg/ml. final medium).
* The numbers preceding ' A ' denote concentration expressed as i.u./ml. of final medium.
All explants were fixed after 4-14 days, a few in buffered 2 % osmic acid for
1 /i sections stained with toluidine blue, and the remainder in Zenker's fluid for
complete serial paraffin sections at 8/t. The latter were stained (see Hardy,
1967 a) with Mayer's haemalum, eosin and picric acid (HEP), Mayer's haemalum and Alcian blue (HAB), or by the periodic acid Schiff procedure (PAS) in
the presence or absence of diastase digestion and of counterstaining by Mayer's
haemalum.
The explants of skin from the trunk were either devoid of hair follicles
(13^-day embryos) or contained pelage hair follicles in early stages of development (14-, 15-day embryos). Each explant of upper lip in Exps. 2 and 3 contained
all the five rows of mystacial vibrissal follicles from one side of the face (Textfig. 1) and each 'half upper lip' explant in Exp. 4 contained two or three rows.
The lower jaws were bisected medially to provide explants of 'half lower jaw',
of which those in Exp. 2 contained interramal vibrissal follicles while those in
Exp. 4 contained submental follicles of a type intermediate between vibrissal
and pelage.
The use of Maximow slides permitted daily microscopic examination of
Skin response to vitamin A excess
159
living explants and recording of the development stages of many follicles from
day to day. Other follicles, however, were obscured. The stage of the most
advanced follicle which was clearly visible in the living explant each day was
arbitrarily chosen to record the stage of the explant, since there are wide
variations in follicle development times and rates within one skin region both
'A Interramal
Text-fig. 1. Head of mouse showing the main groups of vibrissae.
Table 2. Summary of stages of development of pelage
and vibrissal hair follicles in the mouse
Average age at
earliest appearance
(days from conception)
Pelage follicles
dorsal and
lateral trunk
Stage no.
0
1
2
3a
3b
3c
4
5
6
7
8
No follicles
Follicle placode or plug
Pre-papilla
Dermal papilla invagination
begun (depth < width)
Dermal papilla depth = width
Dermal papilla fully invaginated
(depth > width)
Hair cone
Hair canal
Fully keratinized hair formed
Tip of hair in canal
Tip of hair emerged
<14
14
15
16
Mystacial
follicles
<12
12
13
13*
17
18
14
14
19
20
21
22
23
14|
15
16
17
Modified from Davidson & Hardy (1952).
171
160
M. H. HARDY
in vivo and in vitro. In a fixed explant the stage recorded was that of the most
advanced follicle found in the complete serial sections, and there was good
agreement between observations on living and fixed tissue. Stages 1-8 in hair
follicle development in the mouse as originally defined for the pelage hairs by
Hardy (1949), and a later subdivision of stage 3 (Hardy, 1967b), were used.
Table 2 summarizes the criteria for recognizing these stages and the embryonic
age at which each was first observed in sections of skin from the dorsolateral
trunk (pelage) and the upper lip (vibrissae) respectively. The distinguishing
morphological features of vibrissal follicles at all stages of development and
their faster rate of development have been described previously (Davidson &
Hardy, 1952).
8 7 6 -
1
g
5H
13
\
•o 3c -
S 3b w
3a2 -
1 -
t
0 I
12
I
13
I
14
I
15
I
16
I
17
I
18
I
19
I
20
I
21
I
22
1
23
I
24
I
25
I
26
I
Days from conception
Text-fig. 2. Graph showing rates of development of pelage follicles from 15-day
embryos, x • • • x , in trunk skin in situ; •
• , in explant showing maximum
progress obtained in control medium (Exp. 1); O - - O , in explant showing the
maximum progress obtained in vitamin A medium (25-0 i.u./ml.). The meaning of the
shaded bands in this and subsequent text-figs, is explained in the Discussion, p. 176.
RESULTS
To understand the peculiar orientation of mouse embryonic skin with developing hair follicles in coverslip cultures, the reader should consult earlier papers
(Hardy, 1949, 1951). The overall behaviour of explants and the histology of
the dermis and epidermis in the present series of experiments has already been
described (Hardy, 1967 a).
Skin response to vitamin A excess
161
(1) Pelage hair follicles from the trunk of 15-day embryos (Exp. 1)
At the time of explanation, the skin contained the first-formed pelage follicles
at the beginning of stage 2, evenly spaced over the skin surface. A few less
developed follicles (stage 1) had appeared between them.
Observations on living explants and sections
During the first 4 days in vitro some normal follicle development took place
in all treatment groups, and at least six explants reached stage 4, the normal
stage for 19 days in vivo (Text-fig. 2). Some new follicles were initiated in most
8 -
.x
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Days from conception
Text-fig. 3. Graph showing rate of development of pelage follicles from 13J-day
embryos, x • • • x, in trunk skin in situ; O - - O , in explant (Exp. 2) showing the
maximum progress obtained in vitamin A medium (6-2 i.u./ml.).
explants. Thereafter differences were noted in the living explants between control
and vitamin-treated groups. Because of the curling of the epidermis around the
dermis which frequently occurs in 15-day explants with the culture technique
used, many follicles were prevented by poor nutrition from continuing to
develop at the normal rate. However, in three of the eight control explants
which were cultivated for 8 or more days, normal keratinized hairs were formed
(Text-fig. 2), and six out of eight explants reached stage 4 at least. There was no
development beyond stage 4 in either of the vitamin A-treated groups, and most
of the follicles which could be distinguished in the living explants were seen to
162
M. H. HARDY
regress to earlier stages—3 a, 2 or 1. Many follicles disappeared completely.
After 8 or more days of cultivation, none of the vitamin-treated explants were
as advanced as stage 4. In one the follicles were observed regressing from
stage 4 at 4 days to stage 2 at 8 days and then partially recovering, to stage 3 c
after 11 days (Text-fig. 2).
(2) Pelage hair follicles from the trunk ofYh\-day embryos (Exp. 2)
Two control groups (combined in Table 1, but actually in media containing
plasma from different batches) were compared with four groups receiving
different levels of vitamin A. At the beginning of the experiment there were no
signs of follicle development.
\
8 -
x
\
•—•
7 -
/
6 -
•r
©—o
X
pi
/M
4 -
^
^
*
'"mar
'//
}
3c 3b -
1
3a -
X
2 1 -
X
X/ '
- ^/
0 \
I
12
13
t
I
I
I
1
I
l
I
I
l
|
l
I
I
14
15
16
17
18
19
20
21
22
23
24
25
26
l
Days from conception
Text-fig. 4. Graph showing rates of development of vibrissal follicles from 13J-day
embryo, x • • • x, in upper lip in situ; •
• , in explant of upper lip (Exp. 2)
in control medium; O - - O, in explant of upper lip in vitamin A medium (12-5 i.u./
ml.) (the upper line represents maximum progress and the lower line minimum
progress of those mystacial follicles which were most advanced at the beginning of
the experiment); •
1, in explant of lower jaw (interramal follicles) in control
medium; • - - • , in explant of lower jaw (interramal follicle) in vitamin A medium
(12-5 i.u./ml.).
Observations on living explants and sections
The first follicles did not appear for 3 or 4 days, more slowly than in vivo.
Once they began, some follicles proceeded to develop at about the normal rate,
but when the experiment was terminated at 11 days the most advanced follicles
Skin response to vitamin A excess
163
were still only at stage 5 (Text-fig. 3). The numbers of successful explants in this
experiment were too small for an evaluation of different treatments, but the
following tentative conclusions were drawn: (a) the 37-5 i.u./ml. level of vitamin
A, which was toxic to the skin (Hardy, 1967 a,) was probably also inhibitory to
the hair follicles (e.g. only one explant in four reached stage 2); (b) at all other
levels of vitamin A (25-0, 12-5, 6-2 i.u./ml.) the development of hair follicles,
though limited, was no more limited than in the control explants; (c) the most
advanced hair follicles in the vitamin A-containing media appeared to be
progressing normally (Plate 1, fig. A), except that in the living state they frequently had a swollen appearance or an irregular outline.
(3) Vibrissal follicles in the upper lip of a \3\-day embryo (Exp. 2)
At the beginning of the experiment most of the largest mystacial vibrissal
follicles in the two explants were at stage 3 a (Plate 1, fig. B), but an occasional
one was at stage 3b. Many smaller follicles were at stages 2 and 1. The dermal
papillae of most follicles were still shallow, and the hour-glass shape which is
characteristic of the largest 14-day vibrissal follicles (Plate 1, figs. C, D) had
not yet appeared.
Observations on living explants
After 2 days the control explant of upper lip reached stage 5 (Text-fig. 4) and
the refractile cones of Henle's layer of the inner root sheath were clearly visible.
After 3 days it was at stage 6 and after 6 days at stage 8. The shape of the follicles
was preserved, and well-developed hairs were seen. The vitamin-treated explant
reached stage 4 after 2 days, when the follicles appeared swollen and flaccid.
After 3 days, swellings were noted on the outer root sheaths of a few of the
follicles (stage 3 c) and by 4 days these were well-defined buds. Elongation of
the buds was observed in the remaining 3 days of culture, and the refractile
cones of Henle's layer were not seen in these follicles. Follicle differentiation
remained 2 or 3 days behind that of the control explant, but continued until
fixation at 7 days. Keratinization of the epidermis was delayed by 2 days in the
vitamin-treated explant, but in other respects the structure of the skin appeared
similar in the two cultures.
Observations on histological sections
After 7 days in vitro the control explants contained many relatively thick
(10-12 ft) keratinized hairs in follicles at stage 8 (Plate 2,figs.A-C). The vitamin
A-treated explant had only a few very fine keratinized hairs (maximum diameter
4-5 /*) in follicles at stage 6, although all tissues were healthy and the dimensions
of follicles and root sheaths were similar to those in the control explant. In the
treated explant, three less advanced follicles (two at stage 5 and one at stage 4),
which in the living state had shown lateral budding, were traced through serial
sections. The main part of these follicles and the surrounding tissues were
164
M. H. HARDY
healthy and differentiating normally (Plate 2, figs. D-G). The buds were at first
solid cylindrical outgrowths from the outer root sheath (Plate 2, fig. D) consisting of cuboidal epithelial cells. One had a terminal swelling (Plate 2, fig. G)
and the beginning of a lumen (Plate 2, fig. F).
Several other glandular structures not seen in the living explants which were
receiving excess vitamin A were discovered in the fixed tissue and two were
traced through serial sections. They were both branching epithelial downgrowths from the epidermis. One was mainly solid, with two unusually large
sebaceous glands budding from it. The other had a duct with a long narrow
lumen, a very small sebaceous gland and a solid terminal swelling with many
mitoses. No trace of similar structures was found in the control explant. The
significance of these epidermal appendages will be referred to in the discussion.
(4) Vibrissal follicles in the lower jaw of a \3\-day embryo (Exp. 2)
At 13^- days the three interramal follicles were at stage 1 in a papilla-like
elevation of the skin in the mid-ventral line (Text-fig. 1). The development of two
interramal follicles very close together was observed in the living control explant
consisting of one half of the jaw, and sections after 11 days of cultivation
showed one follicle at stage 5 (Text-fig. 4) and a second follicle base at stage 3 c
joining the side of the first follicle. A third follicle close by, probably a pelage
follicle, was observed first at 9 days and reached stage 3 a at 11 days. In the
vitamin A-treated explant a single follicle, which was probably interramal,
reached stage 2 at 2 days and stage 3 a at 4 days, but when fixed at 11 days was
atrophic and had only reached stage 3 c. A few other atrophic follicles at stages
1 and 2 which were seen only in sections may have been submental vibrissal or
pelage follicles. No budding from follicles was observed in the lower jaw explants.
(5) Vibrissal follicles in the upper lip of 14-day embryos (Exp. 3)
The observations on budding from follicles made in Exp. 2 led to the design
of Exp. 3, in which four pairs of carefully matched explants from the upper lip
and lower jaw were compared. Some of the rapidly enlarging explants were
PLATE 1
Fig. A. Section through explant of trunk skin from 13^-day embryo after 11 days in 6-2 i.u./
ml. vitamin A medium (Exp. 2), showing pelage follicle developed normally to stage 5.
d.p. = dermal papilla; o. = outer root sheath; He. = Henle's layer; Hu. = Huxley's layer;
cu.i. = cuticle of inner root sheath; cu. = cuticle of developing hair; co. = cortex of developing hair (not yet keratinized). HEP, x 250.
Fig. B. Section through upper lip of 13^-day embryo (control to Exp. 2) showing vibrissal
follicle at advanced stage 3 a. Note shallow dome-shaped dermal papilla (d.p.), and beginning
of condensation of mesenchyme cells to form connective tissue sheath (c.t.). HEP, x 250.
Fig. C. Section through upper lip of 14-day embryo (control to Exp. 3) showing vibrissal
follicle at stage 3 b with dermal papilla (d.p.), hour-glass shape and connective tissue sheath
(c.t.). HEP, x 100.
Fig. D. Vibrissal follicle in fig. C at the same magnification as fig. B to show the greater
follicle length and deeper dermal papilla invagination. HEP, x 250.
J. Embryol. exp. Morph., Vol. 19, Part 2
100/i
PLATE 1
100//
100//
100//
C
? „• .- / •
M. H. HARDY
facing p. 164
/. Embryol. exp. Morph., Vol. 19, Part 2
100//
M. H. HARDY
PLATE 2
/. Embryol. exp. Morph., Vol. 19, Part 2
M. H. HARDY
PLATE 3
J. Embryol. exp. Morph., Vol. 19, Part 2
M. H. HARDY
PLATE 4
Skin response to vitamin A excess
165
divided after 4 days to ensure adequate nutrition, so that some groups eventually
contained as many as seven explants.
Immediately after explantation the living mystacial follicles were seen to be
at stages up to 3b, and were a little more advanced than those in the 13^-day
embryos. The larger ones had acquired the characteristic hour-glass shape of
vibrissal follicles (Plate 1, fig. D), and a well-defined connective tissue sheath
(Plate 1, fig. C).
8
7
6 -
g 5
S
o*
4 -
2 3b
S 5a
t /,i
/ " /
-
Metaplasia+regression
b
2
o
o
\
1
0
"I
12
1
13
t
\
e
1
1
1
1
1
1
1
1
I
1
1
1
I
14
15
16
17
18
19
20
21
22
23
24
25
26
I
Days from conception
Text-fig. 5. Graph showing rates of development of vibrissal follicles from 14-day
• , in explants of upper lip (Exp. 3)
embryos, x • • • x, in upper lip in situ; •
in control medium; O - - O, in explants of upper lip in vitamin A medium (12-5 i.u./
ml.) (the upper line represents maximum progress and the lower line minimum
progress of the mystacial follicles which were most advanced at the beginning of the
experiment); •
• , in explants of lower jaw (submental follicles) in control
medium; •
• in explants of lower jaw (submental follicles) in vitamin A
medium (12-5 i.u./ml.).
PLATE 2
Figs. A-C. Sections through explant of upper lip of 13^-day embryo cultivated for 7 days in
control medium (Exp. 2), showing normal vibrissal follicles at stage 8. Figs. B and C illustrate
the middle and lower portions of the same follicle andfig.A the upper portion of a similar
follicle with emerging hair tip (/*.). HEP, x 250.
Figs. D-G. Sections through a single vibrissal follicle in explant of upper lip from the same
embryo as figs. A-C but cultivated for 7 days in 12-5 i.u./ml. vitamin A medium (Exp. 2),
showing gland-like structure arising, from follicle. Note enlarged outer root sheath but
otherwise normal differentiation of follicle layers to stage 5. /. = lumen. HEP, x 250.
166
M. H. HARDY
Observations on living explants
For the first 2 days in culture the vibrissal follicles developed normally
although a little slowly in both treated and control groups (Text-fig. 5). After
3 days many follicles in both groups had reached stage 4, but in one explant in
the vitamin A medium the epidermal parts of the follicles had rounded bases
instead of being invaginated by dermal papillae.
At 5 days all of the five control explants containing epidermis had normally
shaped follicles at stages 4, 5, 6 or 7, but no normal follicles were seen in any
of the corresponding five vitamin-treated explants. Most of the follicles in the
vitamin A group were long and thin, and had lost their dermal papillae, thick
connective tissue sheaths and internal structure. Their epithelial outlines were
irregular and buds were commencing to form from the outer root sheaths. At
7 days the follicles in control explants were still developing normally (stages 6,
7 and 8; Plate 3, figs. A, B), and small sebaceous glands with one to three
differentiated sebaceous cells were observed in two out of five explants. In all
of the four treated explants in which details could be observed clearly, most of
the follicles were quite abnormal, and only one of normal appearance was
observed, at stage 4. The others had no dermal papilla, and knobs or cylinders
of cuboidal cells arose from one or more sites on the outer root sheath (Plate 3,
figs. C, D). The thick connective tissue sheaths were absent from these irregular
follicles. There was also an excessive development of sebaceous cells at this
time. In every treated explant many of the follicles showed buds or parts of the
original follicles which were filled with differentiated sebaceous cells and dark
brown masses of sebum or sebum-like material.
PLATE 3
Fig. A. Entire living unstained explant from upper lip of 14-day embryo after 7 days in control
medium (Exp. 3) showing numerous vibrissal follicles at stages 6-8 attached to central mass
of keratinizing epidermis, d.p. = Dermal papilla; c.t. = connective tissue sheath. x40.
Fig. B. A follicle at stage 8 in the upper part of fig. A. d.p. = margin of dermal papilla;
o. = outer root sheath; He. = Henle's layer; co. = developing hair cortex (not keratinized
at this level in follicle), x 100.
Fig. C. Entire living unstained explant from upper lip of same embryo after 7 days in 12-5 i.u./
ml. vitamin A medium, showing loss of normal structure and budding from follicles, x 40.
Fig. D. A follicle at lower left of fig. C which consisted only of a budding mass of cuboidal
epithelial cells, x 100.
Fig. E. Section through entire explant shown in fig. A after 10 days in control medium, showing
central epidermis and well-developed vibrissal follicles cut at various angles. HAB, x 40.
Fig. F. Enlargement of part of field in fig. E to show follicle structure, d.p. = Dermal papilla;
c.t. = connective tissue sheath; h. = keratinized hair. HAB, x 100.
Fig. G. Section through entire explant shown in fig. C after 10 days in 12-5 i.u./ml. vitamin A
medium, showing central epidermis surrounded by numerous glands and one hair follicle.
HAB, x40.
Fig. H. Enlargement of part of field in fig. G, showing hair follicle at stage 5 (/.) and branching
tubular glands (g.). HAB, x 100.
Skin response to vitamin A excess
167
At 9 days the control explants showed many normal follicles up to stage 8,
but sebaceous glands were no longer seen. Most of the follicles in treated explants
were abnormal, and the single normal follicle previously observed had now
reached stage 7. The buds from other follicles had undergone extensive branching to form complex glandular but mainly non-sebaceous masses. Three out
of four treated explants each contained in addition three or more sebaceous
glands.
At 10 days all control explants showed a number of normally keratinized
vibrissae, while only one of the vitamin-treated explants showed a follicle at
stage 8 (Text-fig. 5). Sebaceous glands were not identified with certainty in any
control explant, but were present in three out of four treated explants. The
branching non-sebaceous glandular masses derived from follicles were seen in
all treated but no control explants.
Some explants were fixed after 10 days and the remainder examined at 12
and 14 days. The two control tissues changed little during the remaining period
of cultivation but the two vitamin-treated explants showed further proliferation
PLATE 4
Fig. A. Section through explant of upper lip from 14-day embryo after 14 days in 12-5 i.u./
ml. vitamin A (Exp. 3), showing large healthy vibrissal follicle (stage 8) cut obliquely and
containing a minute keratinized hair (/*.). Attached to follicle is a branching tubular gland
with cystic enlargement of some ducts. HAB, x 67.
Fig. B. Section through another part of explant shown in fig. A with parts of two branching
tubular glands. Gland on left has a duct opening directly to epidermis. Gland on right has,
attached to its duct, an epidermal mass (/.) representing the original hair follicle. HAB, x 67.
Fig. C. Section through same explant as in figs. A and B, showing two glandular trees. No
remnants of the original follicle were found in association with the gland on left. HAB, x 67.
Fig. D. Section through another explant of upper lip from 14-day embryo after 10 days in
12-5 i.u./ml. vitamin A medium (Exp. 3) showing four former hair follicles whose complete
transformation into glands was observed in the living state. At left, left centre and right
centre are three branching tubular glands derived from three follicles, and at the upper right
is the sebaceous gland portion of a glandular structure derived from the fourth follicle.
HEP, x67.
Fig. E. Section through explant of upper lip from 14-day embryo after 10 days in 12-5 i.u./ml.
vitamin A medium (Exp. 3) showing differentiated sebaceous cells (s.c.) at left, and sebaceous
cells breaking down to produce sebum (se.) at right. HEP, x 250.
Fig. F. Another part of explant shown in figs. A-C showing transverse sections of a large
cyst and several small ducts (d.) and terminal portions of branching tubular glands. HEP,
x250.
Fig. G. Part of section seen in fig. D enlarged to show oblique section of small duct (on left)
and two terminal swellings with small lumen (/.) surrounded by cuboidal (?'alveolar') cells.
HEP, x250.
Fig. H. Another part of explant shown in figs. A-C and F with small gland ducts (d.) and
'alveoli', the latter with a very small lumen (/.) of an intercellular canal. HAB, x 250.
Fig. I. Duct of tubular gland in same explant showing mucopolysaccharide reaction. The dark
shade of the cytoplasm of marked cell (c.) and of strands crossing the duct from this cell are
due entirely to Alcian blue staining. HAB, x 1000.
168
M. H. HARDY
of the non-sebaceous glands, and traces of patent ducts and terminal 'alveoli'.
The sebaceous secretion was also still very evident. The hair follicle in the
treated explant which was previously observed at stage 8 was still visible, and
two other follicles in the same explant reached stage 5. A second treated explant
now had several normal-looking follicles at stage 4. Some of these 'recovering'
follicles had acquired a fairly well developed connective tissue sheath, but still
had an irregular epithelial outline and lacked the typical hour-glass shape.
Observations on histological sections
Three of the four control explants fixed after 10 days and the two fixed at
14 days showed essentially normal vibrissal follicles at stage 8, with relatively
thick and normally keratinized hairs (Plate 3, figs. E, F). Some assumed distorted shapes, probably due to abnormal tensions in the explants, but none
showed budding from the outer root sheath or abnormal gland formation. The
sebaceous glands, which in vivo remain very small in vibrissal follicles, had
disappeared from these explants. In each follicle the dermal papilla remained
fully invaginated and was usually healthy and the thick connective tissue sheath
was still present (Plate 3, fig. F). The fourth control explant fixed at 10 days
was derived by subdivision at 4 days when it consisted of epidermis and dermis
with no hair follicles. Newly initiated follicles, which were observed in the
living explant 3 days later (i.e. after 7 days in vitro), were at stage 3 b at 10 days,
and also appeared normal. The latter were probably pelage follicles, although
it was not possible to establish this from morphological criteria. The sections
stained in HAB showed alcian-positive reactions only in the locations where
they are found regularly in normal active hair follicles in vivo (Braun-Falco,
1958).
By contrast, the vitamin-treated explants sectioned after 10 and 14 days
showed a wide spectrum of hair follicle and gland differentiation. Follicles
were found in various stages of departure from normal (Plate 3, figs. G, H;
Text-fig. 5), and only one explant contained hairs. The three minute hair tips
were found in follicles which had normal dermal papillae and root sheaths, and
also some large sebaceous glands and extensively branching tubular glands
(Plate 4, fig. A). Several other hair follicles had normal inner root sheath cones,
sebaceous glands and branching tubular glands, but no hairs. Some had both
types of glands and shrunken dermal papillae, but no root sheaths. The majority
of follicles originally present were represented only by a solid downgrowth
from the epidermis, from which the sebaceous and non-sebaceous glands were
derived (Plate 4, fig. B), but some were lacking even this remnant of the original
structure (Plate 4, fig. C). In two explants all the follicles originally present were
completely transformed into sebaceous glands and tubular branching glands
(Plate 4, fig. D ; Text-fig. 5). There were also a few small follicles which, like
those in a control explant referred to above, were initiated in vivo, and were
developing normally up to stage 4 without any buds, but all the vibrissal
Skin response to vitamin A excess
169
follicles originally present showed some deviations from the behaviour of those
in control explants.
Nearly all the vitamin-treated explants had many differentiated sebaceous
cells and some sebum-like secretory masses produced by the characteristic
holocrine process (Plate 4, fig. E).
The non-sebaceous branching tubular glands were the most prominent
feature of the vitamin-treated explants, and in many instances by repeated
branching they grew as large as the follicles in control medium. In nearly all
explants there were some patent ducts opening either directly on to the surface
of the epidermis (Plate 4, fig. B), or leading to a cyst (Plate 4, fig. F) which
usually communicated by another duct with the skin surface. In the glands which
were best differentiated the collecting ducts had a wide lumen and a two-layered
wall of compact cuboidal or columnar cells with a smooth inner border (Plate
4, fig. B). Terminal bars and terminal webs were recognized in some ducts. The
cysts (Plate 4, fig. F) were lined for the most part by a single layer of flattened
cells which sometimes had an irregular border. The smaller ducts derived by
branching had a single layer of cuboidal cells with a smooth border (Plate 4,
fig. F). Most of the glands had some terminal swellings or 'alveoli' containing
cuboidal cells (Plate 4, fig. G) which could sometimes be distinguished by paler
nuclei and a more eosinophilic cytoplasm. The nuclei were central or slightly
basal. Many terminal swellings did not show any central lumen in the rather
thick sections but the majority had a few very small and irregular intercellular
canals (Plate 4, figs. G, H). Although most of the cells in terminal swellings did
not stain with Alcian blue, both of the explants grown for 14 days had occasional
cells of the same morphological type whose cytoplasm was filled with Alcianpositive and therefore presumably mucous granules. One of the explants fixed
after 10 days had Alcian blue staining material in some lining cells and in the
lumen of several ducts (Plate 4, fig. I).
(6) Vibrissal follicles in the lower jaw of 14-day embryos {Exp. 3)
The submental group of follicles, of intermediate type between pelage and
vibrissal follicles, were at stage 1 or stage 2 when the experiment began. Conditions were not very favourable for the growth of hair folhcles in the lower
jaw explants, but there were some indications of a difference between control and
treated groups. In the control group, follicles were observed at various stages in
living explants (Text-fig. 5), and after fixation at 10 or 14 days, eight normal
follicles at stage 8 were found in the sections from two explants and some
follicles at earlier stages in the sections from two more. In the vitamin-treated
group, follicles were identified in only one of the living explants, and after 10
or 14 days only one normal follicle at stage 8 and a few other follicles at earlier
stages were found in sections from three explants. There was no sign of abnormal
budding or gland development from these follicles.
170
M. H. HARDY
(7) Vibrissalfollicles in the upper lip of\5-day embryos {Exp. 4)
Each explant contained two or three rows of follicles of which the most
advanced was at stage 3 c, 4 or 5 and the least advanced at stage 1 (Text-fig. 6).
Some of the follicles at stage 5 had a small swelling at the side to mark the
position of the future sebaceous gland, although differentiated sebaceous cells
do not appear in the embryo until about a day later, when the hairs begin to
form.
12
13 14 15 16 17 18 19 20 21 22 23 24 25
Days from conception
Text-fig. 6. Graph showing rates of development of vibrissal follicles from 15-day
• , in explants of upper lip (Exp. 4)
embryos, x • • • x , in upper lip in situ; •
in control medium (initially most advanced follicles); O — O, in explants of upper
lip in vitamin A medium (12-5 i.u./ml.) (initially most advanced follicles); A
A,
in explants of upper lip in control medium (initially least advanced follicles);
A - - A , in explants of upper lip in vitamin A medium (12-5 i.u./ml.) (initially
least advanced follicles).
Observations on living explants
After 3 days many follicles identified in the original explants had progressed
to stage 6, 7 or 8 (Text-fig. 6), and the extent of progress was similar in all
treatment groups. Sebaceous cells were recognized in about half of the explants.
After 4 days there was further development of hair follicles in all groups, and
more differentiated sebaceous cells were observed. Although some effects of
vitamin A on the epidermis were apparent at 3 and 4 days (Hardy, 1967 a), no
significant difference was found between treatment groups with respect to hair
Skin response to vitamin A excess
111
follicles. At 6 days, however, some differences showed up in the follicles. In the
control group all 6 explants had reached stage 8 in follicle development and in
the hydrocortisone (HC) group five out of six explants were at this stage, but in
the vitamin A group only one explant and in the A + HC group only three explants out of six were at stage 8. Some follicles in one explant in the A group
had lost their well-defined refractile Henle's layer. The number of keratinized
hairs observed after 6 and 7 days in vitro in the two groups receiving vitamin A
was only about half as great as in the control groups, while the number in the
HC group was greater.
Table 3. Numbers of keratinized hairs observed in explants
of upper lip of 15-day embryos (Exp. 4)
Additions to basic medium
None
No. of keratinized hairs visible in six living explants after
6 days in vitro
No. of keratinized hairs visible in the same living
explants after 7 days in vitro
No. of keratinized hairs in complete serial sections from
the same explants fixed after 7 or 11 days in vitro
No. of above hairs which were perfectly keratinized
Percentage of perfectly keratinized hairs
A A,HC
HC
29
16
15
39
29
18
17
48
77
58
62
80
50
13
12
35
65% 22% 19% 44%
A = vitamin A; HC = hydrocortisone.
Observations on histological sections
Explants sectioned after 7 and 11 days revealed additional differences between
the treated and control groups. While the majority of explants in all groups had
some follicles which had reached stage 7 or 8, there were differences in the
numbers and the quality of hairs produced. Table 3 shows that each of the two
groups receiving vitamin A had about three-quarters as many stage 8 follicles
as the control group, but only one-third as many perfectly keratinized fibres
(which stain bright yellow in HEP and remain unstained in HAB). The remaining hairs in all groups were pink, or pink and yellow, in HEP, and deep turquoise blue in HAB, and showed an unusual amount of bending, irregular
outlines, imperfect cuticular scales, swollen hair cortex, loss of birefringence or
some combination of these features denoting faulty keratinization, even though
many of them had emerged beyond the skin surface and grown considerably
in length.
The four treatment groups were similar with respect to the development of
sebaceous glands. Differentiated sebaceous cells were identified in sections from
one, two or three of the six explants in each group.
Although the formation of non-sebaceous glandular buds from hair follicles
172
M. H. HARDY
was not nearly as extensive in the 15-day upper lip skin as in the 14-day skin,
there were indications that the same tendency to gland formation existed. In the
vitamin A group, three of the four explants fixed after 11 days showed budding
from a few of the smallest and least advanced vibrissal follicles (Text-fig. 6).
One explant had a well-developed branching gland-type structure with a solid
terminal bud, and a duct with a wall three cells thick and a lumen opening at
the skin surface. A second explant had three budding structures derived from
hair follicles which were identified in living explants—(a) a solid downgrowth
from the epidermis with several buds but no dermal papilla, (b) a solid downgrowth with some sebaceous secretion, and (c) a bilobed gland with a duct lumen
opening at the skin surface and the beginning of lumina in the two lobes
(Text-fig. 6). The third explant had a follicle, whose dermal papilla had been
lost, with a lateral bud containing some sebaceous secretion. In the A + HC
group one of the four explants fixed after 11 days had a follicle with an elongated
lateral bud, and a second explant had buds beginning to form from an outer
root sheath. Neither the control group nor the one receiving hydrocortisone
alone showed any of these budding or glandular structures, and all of their
vibrissal follicles differentiated in a more normal fashion (Text-fig. 6).
DISCUSSION
Metaplasia of vibrissal follicles
The most radical changes in response to vitamin A occurred in the vibrissal
follicles of the upper lip in Exp. 3. This appears to be a true metaplasia, in which
cells of the outer root sheath or less differentiated follicle cells changed the
direction of their development and formed branching tubular glands. Sometimes
an entire hair follicle was transformed into an entire gland, and it seemed that
most of the follicle cells of epidermal origin were involved, so that a potentially
complex keratin-forming organ changed into an equally complex secretory type
of organ. Other hair follicles were able to pursue the two developmental pathways simultaneously, and produce both a keratinized hair and a secretory-type
gland. In Exps. 2 and 4 a few follicles from the upper lip underwent similar
changes, and it is possible that the glandular structures which were attached
directly to the epidermis in these experiments were also derived originally from
hair follicle rudiments.
The exact nature of the glands formed remains an open question. They are
morphologically distinct from sebaceous glands, which are usually simple
saccular glands in the mouse, and even in their more elaborate forms such as
the human Meibomian glands are branched saccular but not tubular glands.
Furthermore, the metaplastic glands were not undergoing holocrine secretion
or producing fatty substances. They could also be distinguished from the eccrine
sweat glands of mammals, which are unbranched coiled tubular glands arising
from the epidermis independently, and which are absent from the hair-bearing
Skin response to vitamin A excess
173
areas in the mouse. They differ also from the apocrine sweat glands, the unbranched coiled tubular glands with wide terminal lumina which are attached
to the hair follicles in many species but not in the mouse. In species possessing
apocrine sweat glands a single gland begins as a bud from the ental side of a hair
follicle (i.e. in the obtuse angle between follicle and skin surface) above the neck
of the sebaceous gland. The metaplastic glands, on the other hand, arose by
budding at one or more points from any level of the follicle.
The origin of the metaplastic glands differed in several ways from that of
mammary glands, the only remaining major group of glands derived from the
mammalian epidermis. The mammary glands of the mouse develop from large
rounded epidermal buds after a lag period of about 5 days (Balinsky, 1950<z, b)
with a primary sprout from which secondary and later branches are derived.
Mammary glands are characterized both in vivo and in vitro by early duct lumen
formation and the absence of terminal swellings or alveoli during the early
weeks of development (Hardy, 1950) unless suitable hormones are added to
those already present in the usual biological media (Lasfargues & Murray,
1959).
Caution must be exercised in interpreting the nature of the secretory process
in glands which are obviously immature. Certainly the metaplastic glands
showed no signs of holocrine activity, and, in the absence of any evidence of
apocrine secretion, they would be classified as merocrine or eccrine skin glands.
The cells composing the terminal swellings corresponded morphologically to
the 'special serous' (i.e. non-zymogenic) type discussed by Jacoby & Leeson
(1959), but a few of these cells were filled with mucous granules and thus
resembled the occasional 'mucoserous' cells of human mixed salivary glands
(Bloom & Fawcett, 1962). One may therefore refer to the glandular metaplasia
of hair follicles but there is as yet insufficient evidence for regarding this as a
specifically mucous metaplasia such as is found in chick epidermis (Fell &
Mellanby, 1953).
The vitamin A-induced glands bore a surprising resemblance to some structures of ectodermal but not epidermal origin—the developing salivary glands.
The vitamin A-induced glands were compared with the developing mouse
salivary glands as studied both in vivo and in vitro by Borghese (1950), and with
the salivary glands which developed in the explants of lower jaw in the present
experiments (Hardy, 1967 c). (The metaplastic glands were found only in the
upper lip explants while the salivary glands were found only in the lower jaw
explants, so there was no possibility of confusing them). The metaplastic glands
resembled the submandibular rather than the sublingual glands in their general
aborizing form, the histology of large and small ducts and the development of
cysts from ducts in vitro, the histology of terminal swellings, and even the
occasional appearance of morphologically' serous' but histochemically' mucous'
cells. Since both the metaplastic glands and the salivary glands (Jacoby, 1959;
Jacoby & Leeson, 1959) were still immature when the tissues were fixed, it is
12
JEEM 19
174
M. H. HARDY
not known whether the subsequent differentiation of the two groups of glands
would show a divergence.
Most of the 'specialized' glands in mammalian skin are merely elaborations
of typical sebaceous and sweat glands. However, Lyne, Molyneux, Myktowycz
& Parakkal (1964) found an exception in the chin-gland of the rabbit, which
originated as an apocrine sweat gland from a hair follicle, but resembled the
metaplastic glands of the mouse in two departures from the usual pattern. It
underwent extensive branching to form a system of ducts, and it formed some
eccrine secretory lobules as well as the usual apocrine ones.
Another transformation of hair follicles was described by Dawe (1963) in
mice affected by polyoma virus. The sequence of events could only be inferred
from a series of autopsy specimens, but there were some striking similarities
.to the changes actually observed in the present study in response to vitamin A.
Extensively branching tubular secretory glands and branching sebaceous glands
developed from the side of hair follicles.
Effect of vitamin A excess on sebaceous glands
In Exp. 3 the glandular metaplasia in excess vitamin A was accompanied by
a marked increase in the differentiation and secretion of sebaceous cells, but the
sebaceous glands of control explants either did not form at all or began to
develop and then disappeared. In Exps. 2 and 4 the majority of follicles in
vitamin A-treated groups did not undergo metaplasia and their sebaceous
glands were similar to those in the control groups. However, in the few metaplastic follicles produced by vitamin A excess, sebaceous differentiation and
secretion were also greater than that in the non-metaplastic follicles of the
control group. In all three experiments, differentiated sebaceous cells were
located mostly in the usual position at the upper part of the follicle neck, but
occasionally, like the non-sebaceous buds, they appeared at other levels along
the follicle. Thus it seems that vitamin A may have a stimulating effect on
sebaceous glands, at least under the conditions in which it also produces a
glandular metaplasia of a non-sebaceous type. Sebaceous glands were apparently
stimulated to differentiate and secrete rather than to grow faster or to undergo
branching.
Interference with hair follicle development and hair
differentiation caused by excess vitamin A
A number of effects of vitamin A excess on hair follicles were inhibitory to
the follicles without adversely affecting the rest of the skin. The metaplastic
changes in vibrissal follicles from the upper lip in Exps. 2-4 were all accompanied by some inhibition of normal follicle development. Even the follicles
which achieved some normal development as well as glandular metaplasia
showed a delay in follicle differentiation, and their hairs were either abnormally
fine or absent. The majority of mystacial follicles from 15-day embryos (Exp. 4)
differentiated normally and did not undergo glandular metaplasia, but the large
Skin response to vitamin A excess
175
number of imperfectly keratinized hairs can be attributed to the hypervitaminosis. A small number of observations on vibrissal follicles from the lower jaw in
Exps. 2 and 3 suggested a similar delay or inhibition of follicle development in
the presence of excess vitamin A without any inhibition of epidermal growth or
differentiation. Pelage follicle development also was inhibited in Exp. 1, and
most of the follicles regressed. While the regression in 25-0 i.u./ml. vitamin A
might perhaps be attributed to a toxic effect of the medium, since there were
also a few degenerative changes in the epidermis and dermis (Hardy, 1967 a),
this is unlikely to be the explanation for follicle regression in 12-5 i.u./ml., when
the skin was just as healthy and vigorously growing as that in control medium.
Failure of hydrocortisone to affect hair follicles
Very little change in epidermal differentiation (Hardy, 1967 a) or in hair
follicle development was brought about by adding hydrocortisone to either
control medium or vitamin A medium in Exp. 4. This was unexpected, since
hydrocortisone in similar concentrations antagonized the action of vitamin A
in many organ culture systems (Lasnitzki, 1965), and also when administered
alone affected rat skin in vitro (Weissmann & Fell, 1962). It is necessary to check
the activity of the hydrocortisone preparation and to do further experiments
before drawing any conclusions. The observations are included in this paper
merely to provide complete data on vitamin A effects, since experimental work
had to be discontinued for several years.
The 'recovery' of some follicles affected by vitamin A
Text-figures 2 and 5 and many reported observations on individual explants
suggest that follicles which are severely affected by vitamin A after 4 to 6 days
in culture may later recover some capacity for normal differentiation even though
they are still in vitamin A medium and might have attached metaplastic glands.
One would first suspect that this might be due to a falling off of vitamin A
concentration in the medium, but from published analyses of similar media
(Fell & Mellanby, 1953) it seems unlikely that the concentration would fall
below an effective dose. In Exp. 3, the introduction of fresh vitamin A solutions
on the 9th day failed to check this 'recovery' effect. Furthermore, other vitamin
A responses continued in full force in the same explants (Hardy, 1967 a, c), and
the metaplastic glands, once initiated, did not show any signs of regression
towards the end of the experiments. Probably there is a real recovery of the
abihty of hair follicles to differentiate normally after some time in the high
vitamin medium.
Is there a critical period in follicle development for the
metaplastic action of vitamin A ?
It was surprising that the extensive metaplasia of mystacial follicles produced
by vitamin A which was so widespread in all the 14-day explants was less
176
M. H. HARDY
common in a 13^-day explant, infrequent in the 15-day explants and absent
from the vibrissal follicles of the lower jaw. Perhaps there is not only a critical
time interval between the administration of vitamin A and the appearance of a
morphological change, but also a critical period in follicle development at
which the metaplastic action of vitamin A can operate. In many investigations
by Fell and her colleagues (see Fell & Rinaldini, 1965) and in the present series
of experiments (Hardy, 1967 a, c) most of the histological changes in skin and
other epithelial tissues began after 3 or 4 days of vitamin A treatment. Whatever
the earlier critical time and critical stage may be for the significant changes at
the biochemical level, what the histologist observes is a budding from vibrissal
follicles if they are at the later dermal papilla or early hair cone stage (stages 3 c,
4) after about 3 days in excess vitamin A. To illustrate the hypothesis of a
critical stage, in Text-figs. 2-6 the supposed 'histological critical stage' and
'histological critical time' are indicated by shaded bands. The majority of
mystacial follicles from 14-day embryos reached stage 3 c or 4 at the critical
time (Text-fig. 5), and so metaplasia was widespread. New follicles (not indicated
in the figure) arising in vitro many days later were unaffected. The submental
follicles from 14-day embryos treated with vitamin A reached stage 4 several
days too late and did not undergo metaplasia. The largest mystacial follicles
from a 13^-day embryo for technical reasons developed more rapidly than those
from 14-day embryos and reached stage 4 just before the critical time (Textfig. 4). These were not greatly affected, but a few of the next largest follicles
underwent a moderate degree of metaplasia. The majority of mystacial follicles
developed a little later and were unaltered. The interramal follicles, developing
even later, were also unaffected. The largest and medium-sized upper lip follicles
from 15-day embryos were well past stage 4 at the critical time (Text-fig. 6) and
did not show metaplasia, but some of the latest vibrissal follicles to develop in
these explants met the conditions for the metaplastic change.
It is not known whether pelage follicles would undergo metaplasia given the
right conditions, but it may be significant that those which reached stage 4 just
after the postulated critical time (Text-fig. 2) underwent extensive regression,
while those which were not initiated until after the critical time (Text-fig. 3)
proceeded to develop normally. On the other hand, the failure of pelage follicles
to undergo metaplasia may be another instance of the lower responsiveness to
vitamin A of skin on the trunk as compared with skin on the extremities (Hardy,
1967a). This and many other questions, in particular several relating to the role
of the mesenchyme, await further experimentation.
SUMMARY
1. This paper reports the effects of excess vitamin A on the development of
hair follicles in organotypic cultures of embryonic mouse skin. Pieces of trunk
skin, upper lip and lower jaw were cultivated on the surface of a clot of adult
Skin response to vitamin A excess
177
cock plasma and chicken embryo extract in slide preparations for 11-14 days.
Changes in individual living follicles were observed daily and the findings
confirmed from complete serial sections.
2. In skin from the trunk of 13^-day embryos the pelage follicles were
initiated and underwent some normal differentiation, both in the presence and
absence of added vitamin A (6-2-37-5 i.u./ml.).
3. In skin from the trunk of 15-day embryos the pelage follicles differentiated
normally for 4 days in both treated and untreated groups but then regressed in
the vitamin-treated groups (12-5, 25-0 i.u./ml.), while normal differentiation
continued and some keratinized hairs were formed in the control groups.
4. Vibrissal follicles in the upper lip from a 13^-day embryo differentiated
normally for 4 days in excess vitamin A (12-5 i.u./ml.), and then some follicles
became misshapen and developed gland-like lateral buds, while follicles in the
control explant continued normal development and produced many keratinized
hairs.
5. All the vibrissal follicles originally present in four explants of upper lip
from 14-day embryos differentiated normally for about 3 days in excess vitamin
A (12-5 i.u./ml.) and then showed metaplastic changes. Many of them underwent a complete glandular metaplasia, being transformed into compound
tubular glands with branching duct systems, terminal 'alveolar' swellings and
occasional signs of mucous secretion. Some follicles retained traces of the
original structure, and in a few the follicle eventually recovered its capacity for
normal differentiation, producing simultaneously a small keratinized hair and
a vigorously growing gland. The metaplastic glands were unlike any normal
mammalian skin glands but resembled immature salivary glands. At the time of
initiation of the metaplasia there was also an increase in the secretion of sebum
or sebum-like material in the same hair follicles. In the control group without
excess vitamin A the follicles continued normal differentiation and hair production until the experiment was terminated, and there was no metaplasia.
6. Most of the vibrissal follicles in the explants of upper lip from 15-day
embryos continued normal differentiation in the presence of excess vitamin A
(12-5 i.u./ml.), although a higher proportion of the hairs were abnormal in
structure than in the control group. In excess vitamin A a few of the initially
least advanced follicles underwent glandular metaplasia, but no metaplasia
was observed in the control group.
7. Some of the differences between the behaviour of explants from embryos
of different ages could be explained by the hypothesis that there is only a relatively
short critical stage in follicle development during which vitamin A is capable
of inducing metaplasia.
178
M. H. HARDY
RESUME
Metaplasie glandulaire des follicules pileux et autres reactions a un exces
de vitamine A dans des cultures de peau de Rongeur
1. Cet article rapporte les effets d'un exces de vitamine A sur le developpement
des follicules pileux dans des cultures organotypiques de peau embryonnaire de
Souris. Des morceaux de peau du tronc, de la levre superieure et de la machoire
inferieure sont cultives a la surface d'un coagulum de plasma de Coq adulte
et d'extrait embryonnaire de Poulet, pendant l l a 14 jours. Les transformations
dans les follicules vivants sont observees chaque jour et les resultats confirmes
par des coupes seriees completes.
2. Dans la peau du tronc d'embryons de 13,5 jours, les follicules du pelage
apparaissent et commencent une differentiation normale; a la fois en presence
et en absence de vitamine A (6,2-37,5 u.i./ml).
3. Dans la peau du tronc d'embryons de 15 jours, les follicules du pelage se
differencient normalement pendant 4 jours dans les groupes traites et non traites
mais regressent ensuite dans les groupes qui recoivent de la vitamine (12,5, 25,0
u.i./ml), tandis que la differenciation normale continue et que des poils keratinises se forment dans les groupes temoins.
4. Des follicules de vibrisses dans la levre superieure d'un embryon de 13,5
jours se differencient normalement pendant 4 jours en presence d'un exces de
vitamine A (12,5 u.i./ml.), puis certains follicules deviennent malformes et produisent des bourgeons lateraux d'apparence glandulaire; les follicules dans
l'explant temoin poursuivent un developpement normal et produisent de
nombreux poils keratinises.
5. Tous les follicules de vibrisses presents au depart dans 4 explants de levre
superieure d'embryons de 14 jours se differencient normalement pendant environ 3 jours, en presence d'un exces de vitamine A (12,5 u.i./ml.), puis presentent
des alterations metaplasiques. Beaucoup d'entre eux subissent une metaplasie
glandulaire complete, et sont transformers en glandes tubulaires composees,
avec un systeme de canaux ramifies, des gonflements 'alveolaires' terminaux
et a l'occasion des signes de secretion muqueuse. Certains follicules gardent des
traces de leur structure originelle, et quelques-uns recuperent eventuellement
leur capacite de differenciation normale; ceux-ci produisent alors simultanement
un petit poil keratinise et une glande a groissance vigoureuse. Les glandes
metaplasiques ne ressemblent a aucune glande normale de la peau des Mammiferes, mais ressemblent a des glandes salivaires immatures. Au moment ou la
metaplasie apparait, il y a egalement une augmentation de la secretion de sebum,
ou de materiel analogue a du sebum, dans le follicule pileux. Dans le groupe
temoin non soumis a la vitamine A en exces, les follicules continuent la differenciation normale et la formation de poils jusqu'au terme de l'experience et aucune
metaplasie ne se manifeste.
6. La plupart des follicules de vibrisses dans les explants de levre superieure
Skin response to vitamin A excess
179
d'embryons de 15 jours continuent la differentiation normale en presence d'un
exces de vitamine A (12,5 u.i./ml.), pourtant une proportion plus importante
des poils presentent un structure anormale que dans le groupe temoin. Avec un
exces de vitamine A quelques-uns des follicules les moins avances au depart
subissent la metaplasie glandulaire; aucune metaplasie n'apparait dans le
groupe temoin.
7. Certaines des differences de comportement entre les explants provenant
d'embryons d'ages differents pourraient etre expliquees par l'hypothese de la
brievete du stade critique du developpement folliculaire pendant lequel la
vitamine A peut induire la metaplasie.
The author is grateful to Mr Vincent Blancuzzi for careful and patient assistance in the
preparation of thousands of serial sections and to Mrs Joline Spivack and others for technical
help. Dr J. P. W. Gilman of the University of Guelph kindly read the manuscript. Professor
Margaret R. Murray of Columbia University College of Physicians and Surgeons and
Professor Dame Honor Fell, D.B.E., F.R.S., of the Strangeways Research Laboratory gave
encouragement in many ways, and Dr Richard P. Bunge provided in his laboratory an
excellent medium for the growth and differentiation of research scientists. This research was
supported by U.S.P.H.S. N.I.H. Grant NB 04235 and National Multiple Sclerosis Society
Grant 328 administered by Dr Richard P. Bunge.
REFERENCES
B. I. (1950a). On the prenatal growth of the mammary gland rudiment in the
mouse. / . Anat. 84, 227-35.
BALINSKY, B. I. (19506). On the developmental processes in mammary glands and other
epidermal structures. Trans. R. Soc. Edinb. 62, 1-31.
BLOOM, W. & FAWCETT, D. (1962). A Textbook of Histology. London and Philadelphia:
W. B. Saunders.
BORGHESE, E. (1950). The development in vitro of the submandibular and sublingual glands of
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{Manuscript received 8 August 1967)