567
Development 102, 567-574 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
Developmental changes of the proliferative response of mouse
epidermal melanocytes to skin wounding
TOMOHISA HIROBE
Division of Biology, National Institute of Radiological Sciences, Anagawa, Chiba, 260 Japan
Summary
A cut was made on the middorsal skin of mice of
various ages of strain C57BL/10J using fine iridectomy scissors. Specimens from the wounded skins
were fixed at various days after wounding and were
subjected to the dopa reaction and to the combined
dopa-premelanin reaction. When the dorsal skins of
1-5-day-old mice were wounded, the melanocyte population positive to the dopa reaction as well as the
melanoblast-melanocyte population positive to the
combined dopa-premelanin reaction increased dramatically in the epidermis adjacent to a skin wound.
Pigment-producing melanocytes in mitosis were frequently found in the vicinity of a wound immediately
after wounding. When the dorsal skins of 4-5-day-old
mice were wounded, the increase in the melanocyte
and melanoblast-melanocyte populations was smaller
than that of 1-5-day-old mice. The increase in number
of pigment-producing melanocytes in mitosis was
reduced and delayed as compared to 1-5-day-old mice.
When the dorsal skins of 8 5-, 20 5-, and 60 5-day-old
mice were wounded, the increase in the melanocyte
and melanoblast-melanocyte populations was much
smaller than the newborn mice. Moreover, pigmentproducing melanocytes in mitosis were never found.
These results indicate that the proliferative response of
mouse epidermal melanocytes to skin wounding becomes delayed and diminished with development.
Introduction
undergo mitotic division during the healing of skin
wounds (Hirobe, 1983). Mitotic melanocytes were
never found in normal epidermis. These results
indicate that the differentiated melanocytes in the
mouse epidermis can proliferate with or without
external stimuli. However, it is not known whether
the proliferative activity of differentiated melanocytes changes with development. The present study
was designed to solve this problem through the
wounding of the skin during the postnatal development of mice. The response of epidermal melanocytes to skin wounding was analysed by means of light
microscopy using techniques of histochemistry and of
conventional histology.
It has been reported that the number of functioning
melanocytes in the epidermis of mammalian skin is
increased by external stimuli, such as ultraviolet
irradiation (Quevedo & Smith, 1963; Quevedo et al.
1965), carcinogen treatment (Szabo, 1963; Iwata et al.
1981) and skin wounding (Staricco, 1961; Snell, 1963;
Giacometti & Allegra, 1967; Giacometti et al. 1972).
However, whether the increase in the number of
melanocytes is due to differentiation of precursor
melanoblasts or to mitotic division of melanocytes
was not resolved. Recent studies using adult mouse
ear skin show that the epidermal melanocytes are
continuously renewed by mitotic division in normal
circumstances (Rosdahl & Lindstrom, 1980; Rosdahl
& Bagge, 1981) and that the proliferative activity of
epidermal melanocytes is stimulated by ultraviolet
irradiation (Rosdahl & Szabo, 1976, 1978; Rosdahl,
1978). In the dorsal skin of newborn mice, the
pigment-producing melanocytes were shown to
Key words: mouse, melanocyte, proliferation, wounding,
epidermis.
Materials and methods
The animals used in this study were the mouse, Mus
musculus, of strain C57BL/10J (substrain C57BL/lOJHir).
They were given water, fed ad libitum on a commercial diet
(Clea Japan) and maintained at 24±1°C with 40-60%
568
T. Hirobe
relative humidity; 12 h of fluorescent light was provided
daily.
The method of skin wounding was reported previously
(Hirobe, 1983). A full-thickness cut 7 mm long was made
anteroposteriorly on the middorsal skin of mice of 1-5, 4-5,
8-5, 20-5 and 60-5 days of age using fine iridectomy scissors.
The incision extended from the epidermis to the deepest
layer of macrophages under the panniculus carnosus
muscle. Immediately after the incision was made, the
margins retracted and the wound cavity widened to about
2mm. The wounds were not sutured or dressed. The
animals were killed at various times thereafter. The entire
wound area, including the bed, was then removed from the
animals. Biopsy specimens from the wounded skins and
from corresponding fields of skin from intact control
animals were fixed with 16 % formalin in phosphate buffer
(pH7-0) for 20-24 h at 2°C. Each age group was represented by three mice and one sample was obtained from
the wounded skin area of each animal. One wounded skin
consisted of two regenerating epidermes, namely right and
left sides of the wound bed. The experiments were repeated
three times. The specimens were washed with distilled
water and incubated with 0 1 % L-dopa (3,4-dihydroxyphenylalanine, Wako) solution in phosphate buffer
(pH7-4) for 20-24 h at 37°C. This staining reveals tyrosinase-containing differentiated melanocytes (Hirobe, 1982).
The specimens were oriented transversely to the wound
edge and 10 ^m serial sections were deparaffinized and
counterstained with eosin. For combined dopa-premelanin
reaction (combined dopa-ammoniacal silver nitrate staining), deparaffinized sections after the dopa treatment were
incubated with 10 % ammoniacal silver nitrate (Wako)
solution for lOmin at 58°C (Mishima, 1960; Hirobe &
Takeuchi, 1977). This preferential staining reveals undifferentiated melanoblasts that contain unmelanized stage-I and
-II melanosomes in addition to tyrosinase-containing differentiated melanocytes (Mishima, 1964; Hirobe, 1982). The
specimens were also counterstained with eosin.
The number of melanocytes (cells positive to the dopa
reaction) and the number of stage-I and -II melanosomecontaining melanoblasts plus melanocytes (cells positive to
the combined dopa-premelanin reaction) were estimated
per 0-1 mm2 of the epidermis of each section of skin, and
the calculations based on ten consecutive sections with the
width of 1 mm covering the area 0 4 mm2 of the skin.
In some cases, the specimens from wounded and control
animals were fixed with Bouin's fixative and sectioned
transversely to the wound edge. Serial sections, 8^m in
thickness, were stained with haematoxylin and eosin. Pigment-producing melanocytes in resting phase and mitosis
were examined with the light microscope using numerous
sections.
Results
Changes in the melanocyte and melanoblastmelanocyte populations in the epidermis after
wounding
When the dorsal skins of 1-5-day-old mice were
wounded, the melanocyte population positive to the
dopa reaction as well as the melanoblast-melanocyte
population positive to the combined dopa-premelanin reaction increased dramatically in the epidermis
adjacent to a skin wound (Fig. 1A,B)- On day 1 after
wounding, the melanocyte and melanoblast-melanocyte populations in the epidermis within 1 mm of the
wound edge significantly (P<0-05) exceeded the
controls on day 0 and day 1 (Fig. 2). Both populations
showed maximal number on day 3, then gradually
decreased (Fig. 2). From day 3, both populations
were observed in the roots of hair follicles in addition
to the basal layer of epidermis. This suggests that the
epidermal melanoblasts or melanocytes migrate into
hair follicles. In all stages of wound healing, the
number of melanocyte population did not differ
significantly from that of melanoblast-melanocyte
population, suggesting that all melanoblasts differentiate into melanocytes in the epidermis adjacent to a
skin wound. The melanocyte and melanoblast-melanocyte populations appeared in the regenerating
wound epidermis on day 3 and increased in number
(Fig. 2). Both populations were observed in the roots
of the advancing epidermal sheets from day 3 and
lagged behind their forward edges. This suggests that
epidermal melanoblasts or melanocytes increase in
number adjacent to a skin wound and, thereafter,
migrate into the regenerating wound epidermis. Both
populations showed a maximal number on day 7 and
decreased thereafter. From day 7, both populations
were observed in the roots of hair follicles in addition
to the basal layer of the regenerating wound epidermis, suggesting that the epidermal melanoblasts or
melanocytes migrate into hair follicles. In all stages of
wound healing, the melanocyte and melanoblastmelanocyte populations did not differ significantly in
number, suggesting that all melanoblasts differentiate
into melanocytes in the regenerating wound epidermis.
When the dorsal skins of 4-5-day-old mice were
wounded, the increase in the melanocyte (Fig. 1C)
and melanoblast-melanocyte populations (Fig. ID)
in the epidermis adjacent to a skin wound was smaller
Fig. 1. Vertical sections of the dorsal skins of C57BL/10J
mice during wound healing. A cut was made on the
middorsal skins of 1-5- (A,B), 4-5- (C,D), 8-5- (E,F),
20-5- (G,H) and 60-5- (I,J) day-old mice. Cells positive to
the dopa reaction (A,C,E,G,I) as well as to the combined
dopa-premelanin reaction (B,D,F,H,J) are shown in the
dorsal skins on day 3 after wounding. Epidermal
melanocytes or melanoblasts are seen in the vicinity of a
wound (arrows). The number of epidermal melanocytes
or melanoblasts is greater in younger mice (A-D) than in
older mice (E-J). The right sides of all figures indicate
the wound edge and regenerating wound epidermis.
xl20.
Skin wounding and melanocytes
569
570
T. Hirobe
280
| 260
- 240^. 220
= 200.
° 180
.> 160
M
**• T Regenerating
60
•| 1 4 0
9- 120
D. 100
-§ 80
"3 60'
6 40Z
20
0J
0
edge
£o. 4°1
o
o
0'
••—ff
1 2
3
4 5
7
10
Days after wound formation
*
2
3
14
5
7
10
Days after wound formation
E 120
6
I
\ Wound edge
ten 200
O.
O
•O
•>
\
Regenerating
'•5-
I
1 2
3
4 5
7
10
Days after wound formation
14
Fig. 2. Changes in the number of melanocyte positive to
the dopa reaction (A) and melanoblast-melanocyte
positive to the combined dopa-premelanin reaction (B)
in the dorsal skin of l-5-day-old C57BL/10J mice per
0-1 mm2 of the epidermis within 1 mm of the wound edge
( • ) , the control epidermis (D), and the regenerating
wound epidermis (O). Bars indicate S.E.M.
than that of l-5-day-old mice (Fig. 3). In the regenerating wound epidermis, both populations appeared
on day 2, increased in number until day 7, then
gradually decreased (Fig. 3).
When the dorsal skins of 8-5- (Figs 1E,F, 4) or
20-5- (Figs 1G,H, 5) day-old mice were wounded, the
melanocyte and melanoblast-melanocyte populations in the epidermis within 1 mm of the wound
edge exceeded that of the control epidermis. However, the increase in both populations was much
smaller than that of the younger mice. In the regenerating wound epidermis, the melanocyte and melanoblast-melanocyte populations appeared on day 3,
increased in number until day 10, then decreased.
Neither populations exceeded the initial density
(Figs 4, 5).
When the dorsal skins of 60-5-day-old mice were
wounded, no marked increase in the melanocyte and
melanoblast-melanocyte populations in the epidermis adjacent to a skin wound was observed (Figs 1I,J,
14
C57BL/10J
Day 4-5
4? 100
C57BL/10J
Day 1-5
No. of eel Is positiive to (jopa ^
S 260-1
"Si 240
< 220
0
*-..^
o
2 20'
E 280
180160
140
120
o 100
o. 80
60
40
20
0
C57BL/10J
Day 4-5
I 100
C57BL/10J
Day 1-5
\ 1,
80
60
40
20
0
2
3
5
7
10
Days after wound formation
Fig. 3. Changes in the number of melanocyte and
melanoblast-melanocyte populations in the dorsal skin of
4-5-day-old C57BL/10J mice. Data presented as Fig. 2.
6). In the regenerating wound epidermis, the melanocyte and melanoblast-melanocyte populations
slightly increased in number after wounding. Both
populations on day 14 significantly (P<0-05)
exceeded the initial density.
Changes in the mitotic indices of melanocytes in the
epidermis after wounding
When the dorsal skins of l-5-day-old mice were
wounded, melanocytes in mitotic division were found
in the epidermis within 1 mm of the wound edge from
day 1 to day 4 (Figs7A,B> 8). Mitotic melanocytes
were most frequently found on day 2 (Mitotic index = 5-06%; Fig. 8). In contrast, mitotic melanocytes were never found in either the regenerating
wound epidermis or control epidermis.
When the dorsal skins of 4-5-day-old mice were
wounded, mitotic melanocytes were also found in the
epidermis adjacent to a skin wound from day 3 to day
7 (Figs 7C,D, 8). The mitotic index was maximal on
day 5 (0-59 %). The increase in the mitotic indices of
4-5-day-old mice was reduced and delayed as compared to l-5-day-old mice (Fig. 8). In contrast, mitotic melanocytes were never found in either the
regenerating wound epidermis or control epidermis.
14
Skin wounding and melanocytes
16
30
C57BL/10J
Day 8-5
25
1
A
571
C57BL/10J
E 14
6 12
>
>
10
sit
0 20
eel
©
•i 15
8
a.
8.
1 10
o
6
13
No
"S
6
5
0
0
2
3
7
10
Days after wound formation
3
14
14
7
10
Days after wound formation
C57BL/10J
Day 20-5
B
30
C57BL/10J
Day 8-5
T 14
o
<" 12a. 10
o
g. 20
o
I15
!
o
d
Z
a
= 4
o
o 2
6
0J»
5
n
6
•5
I 10
2
3
7
10
Days after wound formation
14
Fig. 4. Changes in the number of melanocyte (A) and
melanoblast-melanocyte (B). Populations of the dorsal
skin of 8-5-day-old C57BL/10J mice. Data presented as
Fig. 2.
i
3
7
10
Days after wound formation
Fig. 5. Changes in the number of melanocyte (A) and
melanoblast-melanocyte (B). Populations of the dorsal
skin of 20-5-day-old C57BL/10J mice. Data presented as
Fig. 2.
e 5
When the dorsal skins of 8-5-, 20-5- or 60-5-day-old
mice were wounded, mitotic melanocytes were never
found in the epidermis within 1 mm of the wound
edge, the regenerating wound epidermis, and control
epidermis (Fig. 8).
14
C57BL/10J
Day 60-5
6 4
.
Discussion
o
}
Regenerating
3
7
10
Days after wound formation
3
7
10
Days after wound formation
The present study demonstrated that the proliferative
response of mouse epidermal melanocytes to skin
wounding was diminished as developmental age advanced, since the increase in the melanocyte and
melanoblast-melanocyte populations as well as the
increase in the mitotic indices of pigment-producing
melanocytes of older mice was reduced and delayed
Fig. 6. Changes in the number of melanocyte (A) and
melanoblast-melanocyte (B). Populations of the dorsal
skin of 60-5-day-old C57BL/10J mice. Data presented as
Fig. 2.
I
14
572
T. Hirobe
Fig. 7. Vertical sections of the dorsal skins of C57BL/10J mice on day 2 (A), day 3 (B), day 5 (C) and day 7 (D) after
wounding. A cut was made on the middorsal skin of 1-5- (A,B) and 4-5- (C,D) day-old mice. Melanocytes in metaphase
are observed in the epidermis (arrows) adjacent to a skin wound. All specimens were fixed with Bouin's fixative and
stained with haematoxylin and eosin. No dopa or silver treatment. x460.
as compared to newborn mice. It has been reported
that melanocytes begin to differentiate in the epidermis around the time of birth and increase in number
until 4 days, then gradually decrease and disappear by
30 days of age in the dorsal skin of C57BL/10J strain
mice (Hirobe & Takeuchi, 1977; Hirobe, 1984).
Therefore, it is conceivable that the differentiating
melanocytes or newly differentiated melanocytes
which can be stimulated to undergo mitosis by skin
wounding diminish in the epidermis after birth. They
are thought to migrate into hair follicle in early days
after birth. These potent cells, which are considered
as 'stem cell', may undergo mitosis by external stimuli
Fig. 8. Developmental change of the mitotic indices of
epidermal melanocytes of the dorsal skin of C57BL/10J
mice stimulated by skin wounding. A cut was made on
the middorsal skin of 1-5- (O), 4-5- ( • ) , and 8-5- (D)
day-old mice (arrows). Specimens were fixed at various
days after wounding with Bouin's fixative. Mitotic indices
of melanocytes in the epidermis within 1 mm of the
wound edge are shown. When the dorsal skins of 20-5-,
and 60-5-day-old mice were wounded, no melanocytes in
mitosis were found.
even after migrating into hair bulbs. This hypothesis
is supported by the observations of Silver et al. (1969)
that the melanocytes in the hair follicle began to
divide when the hair growth cycle entered into new
activation stage (Anagen; Dry, 1926).
In the epidermis of hairy skin, epidermal melanocytes are found only during the early weeks after birth
(Quevedo et al. 1966; Takeuchi, 1968; Hirobe &
C57BL/10J
Day 1-5
2 1
0J
Day 4-5
0 1-5
4-5
8-5
14-5
Days after birth
D a y 8.5
18-5
22-5
Skin wounding and melanocytes
Takeuchi, 1977). However, in the glabrous skin of
ear, nose and tail, numerous differentiated melanocytes are found in the epidermis of adult mice. Recent
studies using the mouse ear skin show that the
epidermal melanocyte is normally in continuous renewal (Rosdahl et al. 1980, 1981). However, it is not
known whether the mitotic activity of epidermal
melanocytes in the mouse skin changes with development. Also, the epidermal melanocytes of adult
human skin are reported to undergo mitotic division
normally (Jimbow et al. 1975). However, it is not
clear whether the mitotic activity of human melanocytes changes from newborn to adult. The solution of
these problems is expected to clarify the mechanism
of the proliferation of mammalian epidermal melanocytes during development.
Bucher et al. (1964) showed that in a regenerating
rat liver DNA synthesis became reduced and delayed
as developmental age advanced. In the postnatal
development of mouse seminal vesicle, the proliferative activity of seminal vesicle cells was shown to be
maximal at both 8 and 30 days of age (Okamoto et al.
1982). Shirasawa & Yoshimura (1982) reported that
the mitotic rate of GH and prolactin cells increased
from 5 to 70 days of age in male rat pituitary. In
contrast, ACTH- and TSH-cells decreased with age.
On the other hand, Takahashi et al. (1984) reported
that the mitotic indices of prolactin cells decreased
steadily from 20 days of age in male rat pituitary,
while the mitotic rate in female rat was maximal at 60
days of age and then decreased. These studies
together with the present study show that the differentiated cells in mammals can undergo mitosis normally or by external stimuli and that the proliferative
activity of mammalian cells changes during development. However, it remains to be solved in a future
study whatever common mechanism exists in regulating the proliferative activity of mammalian cells in
development.
This work was supported in part by Grant 58740290 from
the Ministry of Education, Japan.
573
rhesus monkeys. In Pigmentation: Its Genesis and
Biologic Control (ed. V. Riley), pp. 451-459. New
York: Appleton-Century-Crofts.
HIROBE, T. (1982). Origin of melanosome structures and
cytochemical localizations of tyrosinase activity in
differentiating epidermal melanocytes of newborn
mouse skin. /. exp. Zool. 224, 355-363.
HIROBE, T. (1983). Proliferation of epidermal melanocytes
during the healing of skin wounds in newborn mice.
J. exp. Zool. 227, 423-431.
HIROBE, T. (1984). Histochemical survey of the
distribution of the epidermal melanoblasts and
melanocytes in the mouse during fetal and postnatal
periods. Anat. Rec. 208, 589-594.
HIROBE, T. & TAKEUCHI, T. (1977). Induction of
melanogenesis in the epidermal melanoblasts of
newborn mouse skin by MSH. J. Embryol. exp.
Morph. 37, 79-90.
IWATA, K., INUI, N. & TAKEUCHI, T. (1981). Induction of
active melanocytes in mouse skin by carcinogens: a
new method for detection of skin carcinogens.
Carcinogenesis 2, 589-593.
JIMBOW, K., ROTH, S. I., FITZPATRICK, T. B. & SZABO, G.
(1975). Mitotic activity in non-neoplastic melanocytes
in vivo as determined by histochemical,
autoradiographic, and electron microscope studies.
J. Cell Biol. 66, 663-670.
MISHIMA, Y. (1960). New technic for comprehensive
demonstration of melanin, premelanin, and tyrosinase
sites. Combined dopa-premelanin reaction. J. invest.
Derm. 34, 355-360.
MISHIMA, Y. (1964). Electron microscopic cytochemistry
of melanosomes and mitochondria. J. Histochem.
Cytochem. 12, 784-790.
OKAMOTO, S., TAKATSUKA, D., TATEISHI, K., OGASAWARA,
Y., YAMANE, T., KITAMURA, Y. & MATSUMOTO, K.
(1982). Proliferative pattern of seminal vesicle cells and
the production of testosterone and 5ar-androgens from
birth to adulthood in mice. Endocrinology 111,
1230-1234.
QUEVEDO, W. C , JR & SMITH, J. A. (1963). Studies on
radiation- induced tanning of skin. Ann N. Y. Acad.
Sci. 100, 364-388.
QUEVEDO, W. C , JR, SZABO, G., VIRKS, J. & SINESI, S. J.
References
(1965). Melanocyte populations in UV-irradiated
human skin. J. invest. Derm. 45, 295-298.
BUCHER, N. L. R., SWAFFIELD, M. N. & DITROIA, J. F.
(1964). The influence of age upon the incorporation of
thymidine-2-C14 into the DNA of regenerating rat liver.
Cancer Res. 24, 509-512.
DRY, F. W. (1926). The coat of the mouse (Mus
musculus). J. Genet. 16, 287-340.
GIACOMETTI, L. & ALLEGRA, F. (1967). The effect of
wounding upon the uptake of 3H-thymidine by guineapig epidermal melanocytes. Adv. Biol. Skin 8, 89-95.
GIACOMETTI, L., MONTAGNA, W., BELL, M. & Hu,
F.
(1972). Epidermal melanocyte proliferation in skin
wound healing and after ultraviolet irradiation in
QUEVEDO, W. C , JR, YOULE, M. C , ROVEE, D. T. &
BIENIEKI, T. C. (1966). The developmental fate of
melanocytes in murine skin. In Structure and Control of
the Melanocyte (ed. G. Delia Porta & O. Miihlbock),
pp. 228-241. Berlin: Springer-Verlag.
ROSDAHL, I. K. (1978). Melanocyte mitosis in UVBirradiated mouse skin. Ada derm. Venereol. 58,
217-221.
ROSDAHL, I. & BAGGE, U. (1981). Vital microscopy of
epidermal melanocytes. Ada derm. Venereol. 61,
55-58.
574
T. Hirobe
I. K. & LINDSTROM, S. (1980). Morphology of
epidermal melanocytes in different stages of mitosis.
Ada derm. Venereol. 60, 209-215.
ROSDAHL, I. K. & SZABO, G. (1976). Thymidine labelling
of epidermal melanocytes in UV-irradiated skin. Ada
derm. Venereol. 56, 159-162.
ROSDAHL, I. K. & SZABO, G. (1978). Mitotic activity of
epidermal melanocytes in UV-irradiated mouse skin.
J. invest. Derm. 70, 143-148.
ROSDAHL,
SHIRASAWA, N. & YOSHIMURA, F. (1982).
Immunohistochemical and electron microscopical
studies of mitotic adenohypophysial cells in different
ages of rats. Anat. Embryol. 165, 51-61.
SILVER, A. F., CHASE, H. B. & POTTEN, C. S. (1969).
Melanocyte precursor cells in the hair follicle germ
during the dormant stage (telogen). Experientia 25,
299-301.
R. S. (1963). A study of the melanocytes and
melanin in a healing deep wound. J. Anat. 97, 243-253.
STARICCO, R. G. (1961). Mechanism of migration of the
melanocytes from the hair follicle into the epidermis
following dermabrasion. /. invest. Derm. 36, 99-104.
SZAB6, G. (1963). The effect of carcinogens on
melanocytes. Ann. N.Y. Acad. Sci. 100, 269-278.
TAKAHASHI, S., OKAZAKI, K. & KAWASHIMA, S. (1984).
Mitotic activity of prolactin cells in the pituitary glands
of male and female rats of different ages. Cell Tissue
Res. 235, 497-502.
TAKEUCHI, T. (1968). Genetic analysis of a factor
regulating melanogenesis in the mouse melanocyte.
Jap. J. Genet. 43, 249-256.
SNELL,
(Accepted 25 November 1987)