Veterinary Dermatology 2006, 17, 36 44

Blackwell Publishing Ltd

Mouse epidermal development: effects of

retinoic acid exposure in utero
ROSA A. GARCA-FERNNDEZ*, CLAUDIA PREZ-MARTNEZ, JAVIER ESPINOSA
LVAREZ, ALEX J. DURN NAVARRETE and MARIA J. GARCA-IGLESIAS
*Histology and Pathological Anatomy Section, Department of Animal Medicine and Surgery, Faculty of
Veterinary Science, University of Madrid (UCM), Madrid, Spain, Histology and Pathological Anatomy
Section, Department of Animal Pathology, Animal Medicine, Faculty of Veterinary Science,
University of Len, Len, Spain
(Received 20 May 2005; accepted 24 November 2005)

Abstract Epidermal morphogenesis was studied in vivo following prenatal exposure to retinoic acid (RA). In
pregnant mice, a single oral dose of RA on day 11.5 of gestation failed to induce histological changes in fetal
epidermal development except in epidermal thickness. Epidermal thickness increased from 16.5 days post-coitum
(dpc) onwards, and temporal and spatial epidermal modifications in keratins K5 and K14 related to proliferative
activity of keratinocytes were observed. An RA effect on cell proliferation was supported by a statistically
significant increase in the number of epidermal S-phase cells, containing BrdUincorporated DNA in RAexposed mice compared with nonexposed animals. The prolonged in utero action of RA on epidermal proliferative activity in fetuses and newborns suggests a long-term RA effect that may play a role on the development
and evolution of diseases in adult skin.

I NTRO D U CT ION
Retinoic acid (RA), the most biologically active natural
metabolite of vitamin A, plays a fundamental role in
embryogenesis and in the differentiation of tissues, including the skin, and is involved in the control of epithelial
cell growth.1
RA is used therapeutically for dermatological2 and
neoplastic diseases,3 although the clinical usefulness of
retinoids is limited by their teratogenic potential.46
Postnatal studies on the effects of retinoids in dermatology and oncology have been undertaken in animals7,8
and humans,9,10 following topical application or oral
administration, but knowledge of the in vivo effects on
skin exposed to RA in utero is limited to mouse vibrissal11
and pelage hair follicle12 development. The physiological
retinoid, RA, mediates in a wide variety of activities such
as the proliferation of epithelial cells.13 It plays a role in
the expression of keratin (K) proteins in keratinocytes,14
which express different types of RA receptors.15 These
intermediate filaments have therefore been used as tools
to investigate cutaneous morphogenesis and differentiation.16 Thus, the present aim was to determine the
in vivo effects of prenatal RA exposure on epidermal
development by analysing changes in morphology,
keratin expression and proliferative activity in Navy
Marine Research Institute (NMRI) mice, thereby
complementing previous studies on the effects of this
Correspondence: Dr Rosa Garcia-Fernandez, Histology and Pathological Anatomy Section, Department of Animal Pathology, Animal
Medicine and Surgery, Faculty of Veterinary Science, University
of Madrid (UCM), Madrid, Spain. E-mail: rosaanagf@vet.ucm.es
36

retinoid on vibrissal11 and pelage hair follicles12 and

widening the knowledge of its local, systemic and
temporal actions on the skin.

METHODS
Animals and treatment
Six- to 8-week-old albino NMRI mice (n = 90), weighing
3035 g, were obtained from Antibiticos Laboratories
S.A. (Len, Spain). All were maintained in a cycle of
12 h light and 12 h dark at a controlled temperature
(21 1 C) and relative humidity (55 10%) with free
access to food and water. All experiments were carried
out following the guidelines of the European Law on the
Protection of Animals.17 To obtain timed pregnancies,
individual female mice were placed randomly with
individual male mice at 20.00 h and inspected daily
thereafter at 09.00 h for the appearance of a vaginal
plug, which indicated mating. Appearance of the plug
was designated day 0.5 of pregnancy.
The pregnant mice were treated by oral administration
with 30 mg kg1 body weight of RA (Sigma Chemical
Co., St Louis, MO, USA) in corn oil on day 11.5 of
gestation. This procedure was carried out in the dark
under dim yellow light to retard photodegradation.
Two control groups were established at the same gestation time by treating pregnant mice with an identical
volume of the corn oil vehicle and by leaving one group
untreated. Five embryos across different litters from each
group (RA-exposed, vehicle-exposed and nonexposed)
were obtained daily from the mothers post-mortem
beginning at gestational day 12.5 until day 18.5. In

2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology

Mouse epidermal development after RA exposure

37

Table 1. Primary antibodies used for immunohistochemical labelling

Clone

Character/species

Specificity

Dilution

Source

TROMA 1*
AF 109
AF 138
MK6
MK10
AF 64
ZBU 30

mo/R
po/Rb
po/Rb
po/Rb
po/Rb
po/Rb
mo/M

K8
K1
K5
K6
K10
K14
S-phase cells

ud
1 in 2 105
1 in 105
1 in 105
1 in 4 105
1 in 105
1 in 2 102

R. Kemler
BabCO, CA, USA
BabCO, CA, USA
BabCO, CA, USA
BabCO, CA, USA
BabCO, CA, USA
Zymed Laboratories, San Francisco, CA, USA

mo, monoclonal; po, polyclonal; K, keratin; M, mouse; R, rat; Rb, rabbit; ud, undiluted.
*70% ethanol-fixed tissues fetuses.
L in L (microlitre in microlitre).
Dr R. Kemler, Max Planck Institute, Freiburg, Germany.

addition, three to seven pregnant mice from each

group were allowed to give birth, and their offspring
were euthanized at birth (0) and at 1 day of age. In each
group, five animals from different litters were analysed
for each age.
To assess epidermal cell proliferation, 2 h before
embryo and offspring collection, all pregnant mice and
offspring were given an intraperitoneal injection of 5bromo-2-deoxyuridine (BrdU) (Sigma Chemical Co.)
(1 mg per 20 g body weight in 150 L of saline solution).
BrdU is incorporated into newly synthesized strands of
DNA of S-phase cells in vivo.

Histology
Fetuses and newborn animals were fixed in Bouins
solution or in 70% ethanol and processed routinely to
paraffin wax. Sections (3 m) were cut at three different levels in each instance: one midsagittal and two
parasagittal (one when an eye was seen and the other
when the stomach lumen was observed), and stained
with haematoxylin and eosin (H&E) and Massons
trichrome stains.

Negative and positive control sections for the primary

and secondary antibodies were included. Adult mouse
epidermis was used as positive control for all antikeratin
antibodies except anti-K6 and anti-K8. A hyperplasic
mouse epidermis and mouse hepatic bile ducts were used
as positive controls for anti-K6 and anti-K8 antibodies,
respectively. A mouse previously inoculated with BrdU
was used as positive control for anti-BrdU antibody;
for corresponding negative control sections, the primary
antibody was replaced with Tris-buffered saline (TBS).

Morphometric study: epidermal thickness

From day 12.5 of gestation to 1 day of age, living
epidermal thickness (i.e. without stratum corneum)
was measured in the midsagittal tissue section of each
animal using an ocular micrometre (PK 12.5 m) (C.
Richert, Austria) calibrated with a stage micrometre.
Ten measures on three different sections in each animal
were recorded in each of seven different body regions:
cranium, back, abdomen, thorax, neck, mandible and
muzzle, except in the back where 20 were made.

Epidermal cell kinetics

Immunohistochemistry
From day 12.5 of gestation to 1 day of age, an immunohistochemical study was carried out on epidermis
from the seven body regions cited previously in the
morphometric study. Midsagittal sections, mounted on
poly -lysine-coated slides, were subjected to immunohistochemical staining using the avidinbiotinperoxidase
complex (ABC) method (Peroxidase Standard, Vectastain,
ABC Kit, Vector Laboratories, Burlingame, CA, USA).
Table 1 shows the working dilution and the characteristics of the primary antibodies used. The immunoreactivity of masked antigens was restored by treating
sections with a solution of 0.1% trypsin (Sigma Chemical
Co.) for monoclonal mouse anti-BrdU antibody or with
heat18 for the other antibodies used. After the sequential
incubations with the primary and secondary antibodies and the avidinbiotinperoxidase complex, the
immunoreactivity was demonstrated using diaminobenzidine (DAB, substrate kit for peroxidase, Vector
Laboratories) as chromogen to produce a brown stain
suitable for light microscopy, and the nuclei were
counterstained with haematoxylin.

From day 12.5 of gestation to 1 day of age, the number

of BrdU-positive nuclei per area (m2) was counted in
five 400 fields of the epidermis of a midsagittal tissue
section of each animal using an image analysis system
( version 3.0, Soft Imaging System, Mnster,
Germany). Quantification was undertaken on three
different body regions: cranial (frontal boneatlas length),
dorsal (atlasrump length) and ventral (abdomen
thoraxneck length). Cells that had incorporated
BrdU were identified by the precipitation of a brown
substance in their nuclei.

Statistical analysis

One-way 19 was used for each age (fetuses and

newborns) to test whether the mean of epidermal
thickness and BrdU-positive nuclei per area (m2) of
epidermis differed among the groups examined (RAexposed, vehicle-exposed and nonexposed). As no
significant difference was found between the means
of the two control groups (vehicle-exposed and
nonexposed), the average value of both groups was used
for comparison with the mean of RA-exposed group.

2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology

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RA Garca-Fernndez et al.
Figure 1. Stage 1. Ectoderm: the pattern of
ectodermal keratin expression is shown.

Figure 2. Stage 1: Stratum basale covered by

periderm illustrating the pattern of keratin
expression.

Multifactor 19 was carried out to analyse epidermal

thickness, using body region as covariate. Differences
were considered statistically significant at P < 0.05. Data
were expressed as mean standard error of the mean
(SEM). Analysis was carried out using
version 2.0.

R E SU LTS
Histogenesis and immunohistochemistry
The main histological features of epidermal development could be broadly divided into four stages, and the
sequence did not differ between control and treated
groups.
Stage 1. Twelve and a half to 14.5 days post-coitum
(dpc). During this period, the cranium epidermis was
a single layer of ectoderm (Fig. 1) consisting of flattened
or cuboidal cells that expressed K8 keratin. The control
group expressed K14 at 13.5 and 14.5 dpc and K5 at 14.5
dpc. RA induced an earlier expression of K5 at 13.5 dpc.
In other regions, the epidermis developed into a
stratum basale (SB) covered by periderm (P): a single
layer of cuboidal cells, with clear, spheric nuclei and
scant basophilic cytoplasm covered by a single layer
of flattened cells with dark ovoid nuclei (Fig. 2). In
control animals, the SB expressed at 12.5 dpc, K14 (in
mandible, neck, thorax and back) and K5 (in mandible
and neck). K14 in the muzzle and abdomen, and K5
in the thorax and back first appeared at 13.5 dpc. RA
treatment induced an earlier expression of K5 in the SB
of the thorax and back (at 12.5 dpc) and in the muzzle
and abdomen (at 13.5 dpc). Likewise, an earlier RAinduced K14 expression was observed in the muzzle at
12.5 dpc (Fig. 3).
At 12.5 and 13.5 dpc, the periderm expressed K8 in
both groups but only in the neck and thorax.
Stage 2. Fourteen and a half to 15.5 dpc. During this
period, the epidermis was now developing into a threelayered structure a stratum basale, a stratum intermedium (SI) and an outer periderm. The new layer

Figure 3. Skin (muzzle). RA-exposed fetus. Day 12.5 of gestation.

K14 expression in the stratum basale (SB). Avidinbiotin
peroxidase complex (brown); Harris haematoxylin (blue);
P, periderm; SB, stratum basale; D, dermis. Light microscope:
Olympus Provis AX 70 (Tokyo, Japan).

(the SI) was composed of polygonal cells each with an

ovoid nucleus containing one or two nucleoli (Fig. 4).
The SB in control animals still expressed K5 and
K14 in all body regions. The periderm now contained
K6 as well as K8, and this was more constantly present
from 15.5 dpc onwards. The new SI layer had no keratin
expression at 14.5 dpc, but stained for K1 and K10 at
15.5 dpc.
With RA treatment, the SI of the muzzle, neck and
thorax expressed K1 earlier (14.5 dpc) (Fig. 5, contrast
control group in Fig. 6). Other RA-induced differences
were found in the periderm where K8 expression was
seen in the mandible at 14.5 dpc. K6 was absent from
the neck at 14.5 dpc and the cranium, muzzle, mandible
and back at 15.5 dpc.
Stage 3. Sixteen and a half to 18.5 dpc. Basophilic granules
appeared in the most superficial layer of stratum intermedium from 16.5 dpc onwards, and scattered strands
of stratum corneum (SC) were seen at 18.5 dpc (Fig. 7).
The stratum intermedium was then referred to as the

Figure 4. Stage 2: Stratum basale plus

stratum intermedium plus periderm showing
the pattern of keratin expression.
2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology

Mouse epidermal development after RA exposure

Figure 5. Skin (muzzle). RA-exposed fetus. Day 14.5 of gestation

showing K1 expression in the stratum intermedium. Avidinbiotin
peroxidase complex; Harris haematoxylin. P, periderm; SI, stratum
intermedium; SB, stratum basale; D, dermis.

39

Stage 4. Postnatal 01 day of age. The postnatal epidermis consisted of basal and suprabasal layers, where a
morphological difference between the stratum spinosum
and granulosum was clear, and a stratum corneum was
present (Fig. 8).
As in the prenatal situation, the SS still expressed K1
and K10 and the SB K5 and K14 in all body regions.
Scattered K6 expression in the SS was seen at day 0 of
age (neck, thorax and abdomen) and at 1 day of age
(cranium, neck, thorax, abdomen and back). There was
no expression in the stratum corneum.
RA treatment caused expression of K14 and K5 in
the suprabasal layer (Fig. 9) in addition to the normal
K1 and K10 expression. The first K6-positive suprabasal
cells in RA-exposed mice were seen on 1 day of age in
fewer body regions than in control groups.
In summary, during prenatal development, RA
treatment induced an earlier expression of K5 and
K14 than in controls, and these keratins persisted in
the SS after birth. RA treatment also caused an earlier
expression of K1 and decreased K6 expression in
the SS.

Morphometric study: epidermal thickness

Table 2 shows the mean epidermal thickness in control
and RA-exposed groups. One-way for each body
region revealed a significant epidermal increase at 12.5
dpc (back), 17.5 dpc (cranium, mandible, neck, thorax
and abdomen) and 0 day of age (cranium and thorax),
whereas a significant decrease was shown at 14.5 dpc
(mandible, thorax and back) and 18.5 dpc (back), both
in RA-exposed animals. When body region was used as
covariate (multifactor ), a statistically significant
increase was demonstrated in all skin examined in
RA-exposed mice at 16.5 and 17.5 dpc and 0 day of
age, whereas a significant decrease was only shown at
14.5 dpc.
Figure 6. Skin (muzzle). Control fetus. Day 14.5 of gestation.
No K1 expression in the stratum intermedium. Avidinbiotin
peroxidase complex; Harris haematoxylin. P, periderm;
SI, stratum intermedium; SB, stratum basale; D, dermis.

suprabasal layer or stratum suprabasale (SS). Periderm

was only observed when there was no stratum corneum.
In controls, the SB continued to express K5 and K14
and the SI K1 and K10. Periderm still contained K8
and K6, whereas the stratum corneum always remained
unstained.

Epidermal cell kinetics

More BrdU-positive cells were seen in RA-exposed
animals (Figs 10 and 11), demonstrating an increase in Sphase cells and hence indicating proliferative activity.
The means of BrdU-positive epidermal cells in RAexposed and nonexposed mice were used to compare
the proliferative activity in both groups (Table 3). A
statistically significant increase in proliferative activity
was found in the epidermis from all body regions and
in both the prenatal and the postnatal periods following RA exposure.

Figure 7. Stage 3: Appearance of basophilic

granules in the most superficial layer of the
stratum intermedium (stratum granulosum)
from 16.5 days post-coitum (dpc) onwards
and scattered strands of stratum corneum at
18.5 dpc. The distribution of the layers and
the pattern of keratin expression are shown.
2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology

40

RA Garca-Fernndez et al.

Figure 8. Stage 4: Stratum basale plus

stratum suprabasale plus stratum corneum.
The terminal differentiation of the epidermis
and the pattern of keratin expression at birth
and at 1 day of age are shown.

Figure 9. Skin (back). RA-exposed fetus. Day 1 of age. Note the K14
expression in the basal and suprabasal layers (no expression in
stratum corneum). Avidinbiotinperoxidase complex; Harris
haematoxylin. SC, stratum corneum. SS, stratum corneum;
SB, stratum basale; D, dermis.

DISCU SSIO N
The results show that prenatal exposure of mice to RA
on day 11.5 of gestation did not modify histogenesis
timing but did have a stimulatory effect on the proliferative activity of the developing epidermis. There was
evidence of epidermal hyperplasia mainly at 16.5 and
17.5 dpc and in newborn animals, a feature that has
also been found during topical treatment with RA2022
and previously related to RA hyperproliferative action.22
The finding of a significant increase in the mean number
of epidermal S-phase cells in treated group, as evidenced
by the incorporation of BrdU into their nuclear DNA,
and modifications in the K5/K14 filament system (basal
keratins that are expressed in cells that maintain proliferative capacity)23 provide further evidence of RA
stimulatory action. These findings corroborate previous
descriptions of the proliferative activity of retinoids
in vitro24 and topical treatments.20,25
Epidermis from animals exposed in utero to RA
expressed K5 and K14 proliferation-related keratins

Figure 10. Skin (back). Control fetus. Day 18.5 of gestation. Only a
small number of cells react to anti-BrdU antibody. Avidinbiotin
peroxidase complex; Harris haematoxylin. SC, stratum corneum;
*, granular layer of stratum intermedium; SI, stratum intermedium;
SB, stratum basale; D, dermis.

earlier than nonexposed mice during the fetal period

and, after birth, expressed them in both basal and suprabasal layers of the epidermis. RA-induced migration of
keratinocytes to the superficial layers without loss of
capacity to synthesize keratins related with proliferation has previously been described and attributed to
retinoid interaction with transforming growth factor
beta (TGF).26 However, disturbances in K5 and K14
expression were not observed when RA was applied
topically in normal mouse20 and in human27,28 skin.
The only result that does not seem to support the
conclusion of a stimulatory action of this retinoid on
proliferative activity was the scant K6 expression in
the epidermis from RA-exposed mice. This keratin has
been associated with hyperproliferative processes,29 but
K6 expression is apparently not essential as its absence
in cultured tumour cells is not associated with decreased
cell proliferation.24 Thus, despite the lack of K6 expression, RA-exposure in utero can be considered to induce
a hyperproliferation of the epidermis.
Retinoids are also reported to inhibit epidermal
differentiation.30 Some authors have attributed this to

2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology

Table 2. Comparison of epidermal thickness between NMRI control and RA-exposed mice during the prenatal and postnatal periods

Muzzle
Mandible
Neck
Thorax
Abdomen
Back
All skin examined

Age from birth (days)

Groups

12.5

13.5

14.5

15.5

16.5

17.5

18.5

C
RA
C
RA
C
RA
C
RA
C
RA
C
RA
C
RA
C
RA

11.40 0.85
11.87 1.20
9.13 0.74
9.00 1.05
10.80 0.94
8.30 1.21
8.67 0.89
8.53 1.26
2.73 0.29
3.00 0.46
2.43 0.22
4.10 0.31
7.05 0.22
7.13 0.30

7.95 3.18
2.80 2.84
17.04 4.01
12.40 4.74
13.71 2.96
13.48 3.50
17.44 2.32
15.98 2.75
8.71 3.66
4.05 4.84
9.48 5.08
4.70 7.19
9.05 4.17
4.42 4.93
12.09 3.16
8.95 3.74

6.90 0.31
5.83 0.57
19.44 0.81
16.67 1.41
20.80 0.58
16.75 0.91
27.60 0.80
25.50 1.47
3.71 0.26
2.20 0.47*
12.65 1.04
8.50 1.90
13.55 0.70
8.42 4.93
15.60 0.33
13.19 0.52*

10.06 0.73
9.67 1.27
25.81 0.86
25.38 1.21
24.39 1.05
26.00 1.57
29.79 1.27
29.88 1.68
5.42 0.24
4.85 0.36
25.33 1.85
18.38 2.77
22.00 1.25
18.42 1.27
22.00 0.85
21.63 1.27

20.39 1.77
16.38 2.65
35.86 1.27
34.38 1.68
29.67 1.72
33.38 2.58
31.94 2.15
34.13 2.15
6.72 0.22
7.03 0.33
34.06 2.58
36.88 3.65
32.19 1.41
35.63 2.12
27.81 0.96
33.33 1.44

17.57 2.13
26.38 2.82
33.20 3.95
37.67 6.04
27.46 3.14
41.25 4.15
26.94 2.97
39.50 3.93
6.84 0.22
8.63 0.29
32.70 3.59
46.63 4.75
30.11 3.17
36.19 4.20
27.52 2.99
34.92 3.97*

24.06 0.83
23.44 0.88
38.75 2.09
36.44 2.09
35.28 2.38
35.36 2.69
31.78 2.39
36.92 2.93
7.30 0.27
6.74 0.29
39.64 2.24
36.70 2.65
33.61 1.18
29.22 1.26*
32.76 1.23
30.53 1.31

19.00 1.05
25.17 0.86*
29.00 2.88
37.33 2.35
17.50 8.25
28.83 4.76
15.55 3.42
28.67 2.79
4.87 4.78
12.50 4.78*
31.25 3.59
36.67 2.93
22.10 3.15
27.63 3.64
20.16 2.38
30.18 2.75*

22.67 0.72
25.38 0.63
28.00 2.08
27.13 1.81
22.50 2.33
24.25 1.16
22.50 1.31
22.25 1.13
4.73 0.34
4.95 0.29
29.17 2.63
26.75 3.22
26.17 1.31
24.75 1.14
22.25 1.63
25.71 1.41

Data are expressed in m as mean SEM. C, control group; RA, RA-exposed group; All skin examined, data obtained when body region was used as covariance; , no data. Significantly different from control
group: *P < 0.05, P < 0.01; P < 0.001.

41

2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology

Cranium

Days of gestation

Mouse epidermal development after RA exposure

Body
region

42

RA Garca-Fernndez et al.

Table 3. BrdU-positive nuclei per m2 of epidermis from NMRI control and RA-exposed mice during prenatal and postnatal periods
BrdU-positive nuclei per m2 according to body region
Age (days)
12.5 dpc
13.5 dpc
14.5 dpc
15.5 dpc
16.5 dpc
17.5 dpc
18.5 dpc
0
1

Groups
C
RA
C
RA
C
RA
C
RA
C
RA
C
RA
C
RA
C
RA
C
RA

Cranial

Dorsal
2

1.7 10 1.6 10
8.5 102 1.5 102*
2.5 102 0.5 102
4.2 102 0.4 102*
2.4 102 0.3 102
6.4 102 0.4 102
3.2 102 0.2 102
7.0 102 0.2 102
2.2 102 0.3 102
4.1 102 0.2 102
1.8 102 0.2 102
3.8 102 0.2 102
2.0 102 0.2 102
3.0 102 0.2 102*
0.7 102 0.1 102
1.8 102 0.1 102
1.2 102 0.2 102
2.0 102 0.2 102

Ventral
2

2.6 10 1.2 10
8.3 102 0.6 102
3.3 102 0.5 102
7.1 102 0.4 102
2.3 102 0.5 102
10.2 102 0.6 102
4.3 102 0.3 102
7.9 102 0.3 102
3.0 102 0.3 102
5.1 102 0.2 102
1.9 102 0.2 102
4.1 102 0.2 102
1.9 102 0.2 102
2.9 102 0.2 102
1.2 102 0.1 102
1.7 102 0.1 102*
1.1 102 0.1 102
1.8 102 0.2 102*

2.6 102 1.2 102

14 102 1.5 102
3.5 102 0.8 102
10 102 0.7 102
4.7 102 0.7 102
10.4 102 0.7 102
5.5 102 0.6 102
11 102 0.6 102
3.7 102 0.4 102
6.2 102 0.3 102
3.3 102 0.3 102
6.1 102 0.3 102
3.0 102 0.3 102
4.0 102 0.3 102*
1.7 102 0.2 102
2.7 102 0.2 102
1.8 102 0.2 102
2.8 102 0.3 102*

Data are expressed as mean SEM. dpc, day post-coitum. 0, day of birth; 1, 1 day of age; C, control group; RA, RA-exposed group. Significantly
higher results were found in the RA group at all ages in the three regions studied (*P < 0.05; P < 0.001; P < 0.0001).

of keratinocytes as described previously for in vitro

treatment.36 They also contrast with the absence of an
effect on epidermal differentiation following in vivo RA
treatment in human skin.28,34
In conclusion, RA administered to pregnant mice
induced, in developing skin from their offspring, an
earlier epidermal differentiation. It also stimulated cell
proliferation both in the epidermis and in the pelage hair
follicle.12 Such prolonged RA action on skin proliferation
raises the question as to whether RA-exposure in utero
could induce a long-term effect on adult skin, thereby
playing a role in development, evolution and prognosis
of skin diseases, such as neoplasms, in adult animals.
Longer-term studies of animals exposed to RA in utero
are needed to determine the long-term action of this
retinoid on adult mouse skin carcinogenesis.
Figure 11. Skin (back). RA-exposed fetus. Day 18.5 of gestation.
Many more cells react positively for anti-BrdU than in control
animals. Avidinbiotinperoxidase complex; Harris haematoxylin.
SC, stratum corneum; *, granular layer of stratum intermedium;
SI, stratum intermedium; SB, stratum basale; D, dermis.

RA inhibition of normal regulation of signalling in the

basal layer, associated with RA-induced K6 expression
in epidermis in vivo.24 In this study, reduced K6 expression
following RA treatment indicates that RA does not
have an inhibitory effect on epidermal differentiation.
Changes in keratinocyte maturation have been
demonstrated in previous in vitro studies with RA
inducing a decrease in the expression of K131,32 or K1033
or both.24,28,34 This action has also been observed in
in vivo studies after topical RA treatment of adult
epidermis.31,35 However, in this study, RA induced an
earlier epidermal K1 expression in fetuses. This indicates an earlier RA-induced epidermal differentiation
in contrast to a delay in the terminal differentiation

AC K N OW L E D M E N T S
This work was supported by grants from the Junta de
Castilla y Len (Espaa) (LE04/94). The authors thank
Dr Miguel Angel Vidal Caballero of the Centro de
Investigaciones Biolgicas (Madrid, Spain) for supplying
the anti-K8 antibody and Dr Juan Garca Vieitez of the
Department of Pharmacology, Toxicology and Nursery
(University of Len, Spain) for advice on RA dosage.

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44

RA Garca-Fernndez et al.
Rsum La morphognse pidermique a t tudie in vivo en fonction de lexposition prnatale lacide
rtinoque (RA). Chez les souris gestantes, une seule dose orale de RA au jour 11.5 de la gestation ne provoquait
pas danomalie du dveloppement ftal, sauf au niveau de lpaisseur cutane. Lpaisseur cutane a augmente,
de 16.5 jours post coitum (dpc) et par la suite et des modifications de kratines K5 et K14 relier la prolifration
kratincoytaire ont t observes. Un effet de RA sur la prolifration cellulaire tait confirm par une augmentation significative du nombre de cellules pidermiques en phase S-contenant BrdU incorporated DNA, chez
les souris exposes au RA par rapport aux animaux non exposs. Laction prolonge in utero du RA sur lactivit
prolifrative chez les foetus et les nouveaux ns suggre un effet long terme du RA qui pourrait jouer un rle
sur le dveloppement et lvolution de maladies de la peau de ladulte.
Resumen La morfognesis de la epidermis se estudio in vivo en ratones tras ser expuestos durante el perodo
prenatal a cido retinoico. En ratones gestantes una sola dosis de cido retinoico en el da 11.5 de gestacin no
produjo cambios histolgicos en el desarrollo fetal de la epidermis a excepcin del grosor de la misma. El grosor
de la epidermis aument desde el da 16 postcopula (dpc) en adelante, y se observaron modificaciones espaciales
y temporales en la distribucin de las queratinas K5 y K14, en relacin con la actividad proliferativa de los queratinocitos. El efecto del RA en la proliferacin celular en ratones expuestos a RA comparados con animales no
expuestos fue corroborado por un aumento significativo en el nmero de clulas epidrmicas en fase S (sntesis),
indicado por la presencia de BrdU incorporado en el ADN. La prolongada actividad del RA in utero en la
actividad proliferativa de la epidermis en fetos y neonatos sugiere un efecto prolongado del RA que puede jugar
un papel relevante en el desarrollo y evolucin de enfermedades en la piel de adultos.
Zusammenfassung Die epidermale Morphogenese wurde nach prnataler Exposition zu Retinolsure (RA) in
vivo untersucht. Bei tragenden Musen konnte eine einzige orale Dosis von RA am 11,5. Tag der Trchtigkeit
auer bei der epidermalen Dicke keine histologischen Vernderungen bei der ftalen epidermalen Entwicklung
induzieren. Die epidermale Dicke nahm ab dem 16,5. Tag post coitum (dpc) zu. Es wurden zeitliche und rumliche
epidermale Modifikationen bei den Keratinen K5 und K14 beobachtet, die auf die proliferative Aktivitt der
Keratinozyten zurckzufhren waren. Eine Wirkung von RA auf die Zellproliferation bei RA-exponierten
Musen im Vergleich zu nicht-exponierten Tieren wurde von einer statistisch signifikanten Zunahme der Anzahl
an epidermalen S-Phase Zellen, die BrdU-inkorporierte DNA enthielten, untersttzt. Die anhaltende Wirkung
von RA in utero auf die epidermale proliferative Aktivitt der Ften und Neugeborenen lsst auf eine
Langzeitwirkung von RA schlieen, die eine Rolle spielen knnte bei der Entwicklung und Evolution von
Krankheiten der adulten Haut.

2006 The Authors. Journal compilation 2006 European Society of Veterinary Dermatology