2915
Development 125, 2915-2923 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
DEV3871
Transgene expression of steel factor in the basal layer of epidermis promotes
survival, proliferation, differentiation and migration of melanocyte precursors
Takahiro Kunisada1,*, Hisahiro Yoshida2, Hidetoshi Yamazaki1, Akitomo Miyamoto1, Hiroaki Hemmi1,
Emi Nishimura2, Leonard D. Shultz3, Shin-Ichi Nishikawa2 and Shin-Ichi Hayashi1
1Department of Immunology, School of Life Science, Faculty of Medicine, Tottori University, Nishi-machi
2Department of Molecular Genetics, Faculty of Medicine, Kyoto University, Kyoto 606-01, Japan
3The Jackson Laboratory, Bar Harbor, Maine 04609, USA
86, Yonago 683, Japan
*Author for correspondence (e-mail: [email protected])
Accepted 22 May; published on WWW 9 July 1998
SUMMARY
Mutations at the murine dominant white spotting (KitW) and
steel (MgfSl) loci, encoding c-Kit receptor kinase and its
ligand respectively, exert developmental defects on
hematopoietic cells, melanocytes, germ cells and interstitial
cells of Cajal. The expression patterns of steel factor (SLF)
observed in the skin and gonads suggest that SLF mediates a
migratory or a chemotactic signal for c-Kit-expressing stem
cells (melanocyte precursors and primordial germ cells). By
targeting expression of SLF to epidermal keratinocytes in
mice, we observed extended distribution of melanocytes in a
number of sites including oral epithelium and footpads where
neither melanocytes nor their precursors are normally
detected. In addition, enlarged pigmented spots of KitW and
other spotting mutant mice were observed in the presence of
the SLF transgene. These results provide direct evidence that
INTRODUCTION
The c-Kit receptor tyrosine kinase of the mouse and its ligand,
steel factor (SLF), are encoded by the dominant white spotting
(KitW), hereafter abbreviated as W, and steel (MgfSl), hereafter
abbreviated as Sl, loci, respectively. Mutations in these loci
result in pleiotropic developmental defects affecting pigment
cells, hematopoietic cells, germ cells and interstitial cells of
Cajal in the intestine (Reith and Bernstein, 1991; Williams et
al., 1992; Maeda et al., 1992; Besmer et al., 1993; Galli et al.,
1993; Ward et al., 1994). Most of the defects in these cells are
displayed as a loss of individual cell lineages in homozygous
W or Sl mutant mice and therefore it is hard to delineate what
types of biological processes regulated by c-kit and SLF are
impaired in these cell lineages. For example, the complete loss
of hair pigmentation observed in homozygous W or Sl mutant
mice could be caused by any one of a number of possible events
occurring in melanocytes such as decreased survival, impaired
proliferation, defective differentiation/maturation or impaired
migration. In fact, the biological processes listed above are all
known to be influenced by the stimulation of c-Kit-expressing
target cells by SLF (Galli et al., 1993; Broudy, 1997).
SLF stimulates migration of melanocytes in vivo. We also
present data suggesting that SLF does not simply support
survival and proliferation of melanocytes but also promotes
differentiation of these cells. Unexpectedly, melanocyte stem
cells independent of the c-Kit signal were maintained in the
skin of the SLF transgenic mice. After the elimination of cKit-dependent melanoblasts by function-blocking anti-c-Kit
antibody, these stem cells continued to proliferate and
differentiate into mature melanocytes. These melanoblasts
are able to migrate to cover most of the epidermis after
several months. The SLF transgenic mice described in this
report will be useful in the study of melanocyte biology.
Key words: Kit, steel factor, Melanocyte development, Melanoblast,
Transgenic mouse, c-Kit
Three of the four known targets of Sl and W mutations
originate from highly migratory stem cells. It has been
speculated that the absence of these cell lineages at their final
destination in Sl and W mice may result from inability of their
precursors to migrate properly as well as from their failure to
survive or proliferate (Benett, 1956; Mintz and Russel, 1957).
Although a significant part of the reduction of the number of
germ cells in the mutant embryos is attributed to diminished
proliferation and/or excessive cell death (McCoshen and
McCallion, 1975), it was reported that 30% of We/We germ cells
are found in ectopic sites, suggesting impaired migration of the
mutant germ cells (Bueher et al., 1993). SLF expression along
the migratory pathway of primordial germ cells, from the dorsal
aorta to the genital ridge, and its disappearance after colonization
except in the developing gonad also suggest that a defect in
migratory/chemotactic function occurs in Sl and W mice (Matsui
et al., 1990; Orr-Urtregger et al., 1990; Keshet et al., 1991). A
role of SLF in supporting migration is also suggested for
precursors of melanocytes called melanoblasts. SLF is expressed
in the mesenchyme underlying developing skin of mouse
embryos while melanoblasts migrate dorsolaterally and then
colonize the skin (Matsui et al., 1990; Manova and Bachvarova,
2916 T. Kunisada and others
1991; Wehrle-Haller and Weston, 1995). After melanoblast
colonization in the skin, SLF expression is thought to be
confined to the dermal papillae of hair follicles, where
melanoblasts mature into melanocytes and provide pigment
granules to the hair keratinocytes (Yoshida et al., 1996b).
To further address the migratory/chemotactic roles of SLF,
we have produced transgenic mice expressing SLF in the basal
layer of the skin. In normal mice, the numbers of
epidermal melanoblasts or melanocytes decrease
from day 4 after birth and reach extremely low
levels after one month of age (Hirobe, 1984). A
5′ regulatory sequence of the human cytokeratin
14 (hk14) gene was used to express murine SLF
cDNA in the basal layer of stratified squamous
epithelia including skin, tongue and esophagus
(Vassar et al., 1989). Distribution pattern of
melanoblasts and melanocytes of these transgenic
mice indicated that ectopically expressed SLF
can promote migration of melanocytes in vivo.
This was further supported by genetic crosses of
transgenic mice with W mutant mice. In W/W57
or W57/W57 transgenic progeny, the coats were
mostly pigmented or the size of the pigmented
regions were increased. The increase of the
pigmented region was also observed by crossing
with bt/bt spotting mutant mice.
The effect of SLF on melanoblasts was also
assessed by using the function-blocking anti-cKit monoclonal antibody ACK2 (Nishikawa et
al., 1991; Yoshida et al., 1993). As expected,
ACK2
treatment
completely
prevented
pigmentation of postnatal hk14-SLF transgenic
mice. Unexpectedly, a month after ceasing ACK2
treatment, recovery of skin pigmentation was
observed in the transgenic mice. This clearly
indicates that c-Kit-independent stem cells of
melanocytes, which are present only in the hair
follicles of normal mice (Yoshida et al., 1996a)
are maintained in the interfollicular skin of the
transgenic mice.
collected from (C57BL×SJL)F1 mice as described (Kimura et al.,
1995). Integration of transgenes was verified by PCR of genomic
DNA with transgene-specific primers. The restriction map of the
transgene was described previously (Kunisada et al., 1998).
Genetic crosses
WB/ReJ/C57BL/6J-W/W57 double homozygotes were crossed with
SLFTg1-1 transgenic mice. The W57/+; SLFTg1-1 or W/+; SLFTg1-
MATERIALS AND METHODS
Mice
Original inbred colonies of mice containing the W, W57
or bt mutation were obtained from the Jackson
Laboratory and maintained in our animal colony to
perform genetic crosses with transgenic mice described
in this report. B6 and BDF1 mice were purchased from
Japan SLC Inc. (Shizuoka, Japan). Appropriate pairs
of animals positive for vaginal plugs were scored as 0.5
dpc at noon on that day.
Generation of transgenic animals
Full-length mouse SLF cDNA that could produce both
soluble and membrane-bound forms of SLF (Yasunaga
et al., 1995) was cloned into constructs containing the
human cytokeratin 14 upstream region (Vassar et al.,
1989), rabbit β-globin intron and poly(A) signal of
human cytokeratin 14. Transgene excised from the
plasmid sequence was injected into fertilized oocytes
Fig. 1. Phenotype of hk14-SLF transgenic mice. Transgenic mouse line SLFTg1-1,
heterozygous for the transgene, was used. Newborn(A), 4-day-old (B), 14-day-old
(C) and 2-month-old (D,E) transgenic mice were photographed with their
littermates. Transgenic mice are readily distinguished by their hyper-pigmented area
as shown by arrowheads in A. Arrowheads in E show shaved area of back skin (note
that haired skin of the transgenic mouse is pigmented). Expression of melanocytespecific TRP2 protein was examined by immunohistochemistry for 2-month-old
transgenic skin (F) and littermate (G). TRP2-positive cells (arrowheads) were
detected in the whole epidermis in transgenic skin but were restricted to dermal
papillae in littermates. Asterisks show dermoepidermal junction and an arrow in F
points to a melanin granule responsible for pigmentation of the transgenic skin.
Coat phenotypes of different SLF transgenic animals, SLFTg2-1 (H), SLFTg4-1(I),
SLFTg4-2(J), SLFTg4-3(K), were photographed at their age of 2 month old.
Unpigmented ventral skin region is indicated by an arrow in H.
Development of melanocytes and steel factor 2917
Fig. 2. Effect of ACK2 blocking antibody on the pigmentation of
SLF transgenic mice. 0.5 mg of ACK2 monoclonal antibody was
subcutaneously injected into SLF transgenic and litter mice on days
0, 2 and 4. On day 8 after birth, pigmentation of the skin, including
hair, was completely abolished in transgenic mouse.
1 animals were then crossed with WB/ReJ/C57BL/6J-W/W57 double
homozygotes to obtain W57/W57; SLFTg1-1 or W/W57; SLFTg1-1
transgenic animals and W57/W57 or W/W57 controls. The genotypes of
these W, W57 mutant SLFTg1-1 mice were finally confirmed by the
phenotypes of their offspring. C57BL/6J-bt/bt were crossed with
SLFTg1-1 mice and the resultant bt/+; SLFTg1-1 F1 mice were
crossed with bt/bt to obtain bt/bt; SLFTg1-1 animals.
age, most parts of the skin, including tail, were heavily
pigmented (Fig. 1C). Pigmentation of the unhaired skin such
as mouth and paws (Fig. 1D) as well as haired skin (Fig. 1E)
increased progressively until adulthood. In the skin of SLFTg1
transgenic mice, melanocytes expressing TRP2 protein were
observed in the basal layer of the epidermis including hair
follicles (Fig. 1F, arrowheads). Since TRP2 is the first
melanocyte lineage-specific protein to be expressed in
melanoblasts (Steel et al., 1992; Pavan and Tilghman, 1994;
Wehrle-Haller and Weston, 1995, Kunisada et al., 1996), it
appears that production of SLF in the keratinocytes induced the
proliferation of melanocytes in the transgenic mice. In contrast,
TRP2-positive cells of skin in normal nontransgenic control
mice were restricted to the dermal papillae (Fig. 1G,
arrowheads). Four of the transgenic mice showed slightly
pigmented skin at birth and, in these mice, the entire skin was
heavily pigmented by 4 days after birth (SLFTg2-1 to SLFTg24; the analysis of these mice was reported in Kunisada et al.,
1998). Pigmentation in these mice was apparently more severe
than in SLFTg1 strains. One of these heavily pigmented
strains, SLFTg2-1, showed an unpigmented dorsal region (Fig.
1H, shown by an arrow). All of these severe phenotypes
suffered growth arrest after 2 weeks of age and died within a
few months after birth. A total of 20 F1 of SLFTg2-1 and
SLFTg2-2 mice reproduced by ovary transfer showed similar
phenotype. One transgenic mouse was heavily pigmented in
Histology and immunohistochemistry
Tissues from transgenic and littermate mice were fixed, embedded in
polyester wax and sectioned (7 µm) as described (Yoshida et al.,
1993). After removing wax with ethanol and rehydrating twice with
PBS, the sections were preincubated for 20 minutes in 5% normal
goat serum in PBS and incubated overnight with the primary antibody.
For the primary antibody, rabbit anti-mouse TRP2 polyclonal
antibody provided by V. Hearing (Tsukamoto et al., 1992) diluted
300× with 5% goat serum was used. After three washes in PBS/0.02%
Tween20 (PBST), the sections were incubated with biotin-conjugated
goat anti-rabbit IgG antibody solution (Vector) for 2 hours. After two
washes with PBST and one wash with 0.1 M Tris (pH 8.0), the
sections were incubated with HRP-conjugated avidin (Vector) for 1
hour, washed three times with PBST and developed using a DAB
substrate kit (Vector). Additional sections were incubated with 100×
diluted FITC-conjugated avidin (Sigma) for 1 hour, washed three
times with 0.1 M Tris (pH 8.0) and then mounted for fluorescence
microscopy using Vectashield mounting solution (Vector). Sections
stained with secondary antibody alone were used as a control.
RESULTS
Pigmentation of SLF transgenic mice skin during
postnatal development
Among nine independent transgenic mice expressing SLF
driven by skin-specific cytokeratin 14 promoter, two stably
transmitted a black epidermal color phenotype (SLFTg1-1 and
SLFTg1-2). In these two lines, the overall characters of skin
pigmentation during postnatal development were similar. At
birth nose, pawpads and external genitalia were already
pigmented (Fig. 1A, arrowheads) while most parts of the skin
appeared to be normal. As early as 4 days of age, pigmentation
of the entire epidermis was visible (Fig. 1B). By 14 days of
Fig. 3. Pigmentation of mouth and paws of SLF transgenic mice.
Pigmentation of oral epithelium was noticeable on day 3 after birth
in the transgenic mouse (A) and became more severe on day 11 (D)
whereas no pigmentation was detectable in age-matched normal
littermates (B, 3 days old; C, 11 days old). Paws of forelimbs of 3day-old (E, lower) and 11-day-old (F) transgenic mice. At day 3,
pads were selectively pigmented in the transgenic mouse and no
pigmentation was detected in the littermate (E, upper). Pigmented
areas extended to entire paws at day 11 in the transgenic mouse but
even at this age, pads were not pigmented in the littermate (G).
2918 T. Kunisada and others
the entire skin region at birth and died within 12 hours
(SLFTg3, data not shown). The remaining three transgenic
mice were lightly pigmented in ears, external genitalia and tail,
and not pigmented in the haired skin. In SLFTg4-1, only the
tail was pigmented (Fig. 1I). In SLFTg4-2, tail and external
genitalia were pigmented (Fig. 1J). In SLFTg4-3, tail, external
genitalia and ears, but not haired skin, were pigmented (Fig.
1K). Unless otherwise mentioned, the SLFTg1-1 strain was
used throughout the experiments in this paper.
Hyperpigmentation of transgenic mice is SLF
dependent
Skin-specific expression of SLF from transgene was first tested
by northern analysis (SLFTg1-1, SLFTg1-2 and SLFTg2-1,
data not shown) or RT-PCR with transgene-specific primers
(SLFTg4-1 to SLFTg4-3, data not shown). To confirm that
interfollicular skin pigmentation of the transgenic mice was
dependent on SLF produced by the transgene, we
administrated anti-c-Kit monoclonal antibody (ACK2) to the
newborn mice. ACK2 is known to eliminate proliferating
melanocyte lineage cells by the blockage of SLF induced c-Kit
function (Nishikawa et al., 1991; Yoshida et al., 1993).
Intraperitonial injection of the antibody at 0, 2 and 4 days after
birth completely abolished the pigmentation of normal mice
(compare +/+ control and +/+ ACK2-treated mice of Fig. 2).
This complete depigmentation was also maintained in
antibody-treated SLFTg1-1 mice for at least 1 week after birth
(Fig. 2, tg/+ cont and tg/+ACK2). Thus, increased epidermal
pigmentation in SLF transgenic mice is absolutely dependent
on expression of transgenic SLF.
Extension of the pigmented skin region in SLF
transgenic mice
The appearance of heavily pigmented regions in transgenic
mice such as nose, ears, lip, gums, paws, nails and external
genitalia, which are normally unpigmented or hypopigmented
in the wild-type mice was a common feature of hk14-SLF
transgenic mice as shown in Fig. 1. Among these tissues, nose,
ears, lip and external genitalia were not completely
unpigmented even in the adult wild-type mice: histological
examination of these tissues always indicated the presence of
mature dendritic melanocytes although they were low in
number (data not shown). Therefore, hyperpigmentation in
these regions in SLFTg mice could be readily explained by the
ability of exogenous SLF to promote the c-Kit-dependent
proliferation of melanocyte precursors that had previously
migrated to these areas.
However, in gums, pawpads and nails, we could not detect
mature melanocytes in normal adult mice (data not shown). At
3 days after birth, gums for the incisor teeth and molar teeth of
SLF transgenic mice were heavily pigmented and the entire oral
epithelium including lips was also lightly pigmented (Fig. 3A).
In a wild-type litter, no pigmentation was observed in the entire
region of the oral epithelium although the haired skin region
outside the lips was lightly pigmented (Fig. 3B). At day 11 after
birth, no pigmentation was seen in the oral epithelium of the
wild-type litter (Fig. 3C); however, most of the oral epithelium
including lips and gums was heavily pigmented in SLF
transgenic mice (Fig. 3D). In some cases, the inside of the incisor
tooth was pigmented (data not shown). In paws of transgenic
mice, the pads of the palmar surface of the forelimb were heavily
pigmented at day 3 (Fig. 3E) and the pigmentation over the paws
became extensive, including on the inside of the nails (Fig. 3F).
In contrast, no pigmentation was observed in the unhaired pads
and nails of the wild-type litter (Fig. 3G). These observations
raise the possibility that melanocytes of the transgenic mice
responsible for the pigmentation of the normally unpigmented
areas such as gums or pads were derived from melanocyte
precursors (melanoblasts) that migrated out from the limit of
their normal distribution area where melanocytes in normal mice
eventually reside. Thus, the SLF transgene might have promoted
the further migration of melanocyte lineage cells.
Distribution of melanocyte precursors in SLF
transgenic mice
If extended migration of melanoblasts promoted by exogenous
SLF is responsible for pigmentation of gums and pads of the
transgenic mice, then melanoblasts should be located in those
areas prior to pigmentation in situ. To test this, we performed
immunohistological analysis by using antibody for melanocyte
lineage-specific TRP2 enzyme. In 14.5 dpc embryos, no
melanoblasts stained by anti-TRP2 antibody were found in the
oral epithelium of gums or orpharinx region of either SLF
transgenic or wild-type litter mice (data not shown). In 16.5
dpc embryos, melanoblasts expressing TRP2 were found in the
basal cell layer of the oral epithelium of transgenic mice (Fig.
4A). However, melanoblasts were not detected in the
corresponding part of the wild-type litters (Fig. 4B). After
extensive observation of eight different litters, no melanoblast
was seen in any sections of embryos containing the oral region.
Therefore, we concluded that melanocyte precursors never
reach the mouth epithelium of normal mice. The number of
melanoblasts in the epidermal skin at this stage was
comparable in transgenic and wild-type mice as shown in Fig.
4C and D, respectively. In the newborn transgenic animals, the
number of melanoblasts in the oral epithelium and epidermal
skin was increased significantly (Fig. 4E,F) when compared to
those of 16.5 dpc. Still, no TRP2-positive cells were observed
in normal littermates (Fig. 4G). As expected from the
increasing pigmentation of transgenic mice after birth,
melanoblasts of the newborn transgenic skin outnumbered
those of wild-type littermates (compare Fig. 4F and H). In most
cases, TRP2-positive melanoblasts were found inside of the
primordial molar teeth as indicated by the arrows in Fig. 4E.
In pawpads, TRP2-positive melanoblasts were located in
transgenic 16.5 dpc embryos but no TRP2-positive
melanoblasts were detected in wild-type littermates (data not
shown). Collectively, these data suggest that it is highly likely
that exogenous SLF stimulated the migration of epidermal
melanoblasts of as early as 16.5 dpc embryos to reach the entire
oral epithelium and pads of paws, and then these precursor
cells continued to differentiate into mature melanocytes in situ.
As mentioned in the previous section, mature melanocytes
were observed in the ears of normal mice. By histological
analysis, mature melanocytes were found mostly in the dermis
of the ears of normal mice as shown by an arrowhead in Fig. 4I.
Interestingly, the majority of melanocytes in SLF transgenic
mice were confined to the epidermis as shown in Fig. 4J. As
melanocytes of the ears of normal mice might be maintained by
endogenous SLF expressed in the dermis of the ears (Yoshida et
al., 1996a), movement of the melanocytes of the transgenic mice
to the ear epidermis might be caused by the relatively higher
Development of melanocytes and steel factor 2919
concentration of local SLF derived from the transgene. Also, in
this case, SLF might play a role in the migration of melanoblasts.
Extended coat pigmentation of Sl and W mutant
mice in the presence of hk14-SLF transgene
Melanoblasts originated from the neural crest migrate through a
dorsolateral pathway and cover most of the haired skin region
(Le Douarin, 1982; Hirobe, 1992; Bronner-Frazer, 1993;
Erickson and Perris, 1993; Bennett, 1993; Anderson, 1997).
During this migration phase, melanoblasts are located
exclusively in the dermis and then invade the epidermis around
12 to 13 dpc (Kunisada et al., 1996). Because c-Kit signaling
through SLF is indispensable at least for the survival of
melanoblasts, mutations in the W and Sl loci exhibit reduced or
complete loss of coat color pigmentation. Among these
mutations, W57 showed reduced expression of the c-kit gene of
trunk melanoblasts, giving rise to an irregular white band in the
trunk region as indicated by W57/W57; +/+ in Fig. 5A. When this
mutation is expressed as a compound mutation with W in which
a nonfunctional c-Kit molecule without the transmembrane
region is produced (Hayashi et al., 1991), the amount of c-Kit is
further reduced and results in an almost complete loss of
pigmentation of the hair of the entire body (W/W57; +/+ of Fig.
5A). In the presence of the SLF transgene, the white band of
W57/W57 was greatly reduced in size (W57/W57; tg/+ of Fig. 5A)
and colored skin regions appeared in the head and rump of
W/W57 mice (W/W57; tg/+ of Fig. 5A). In addition, a clear
unpigmented band in the mid-trunk region characteristic of bt/bt
mutant mice (bt/bt; +/+ of Fig. 5B; its responsible gene is not
identified at present, see Mayer and Maltby, 1964) was reduced
in the presence of the transgene (bt/bt; tg/+ of Fig. 5B).
These results could be explained by the stimulation of
migration of melanoblasts as well as stimulation of growth
and/or survival of melanoblasts by the exogenous SLF expressed
in the epidermis. We think that compensation of reduced
melanoblasts by the SLF transgene occurred 14.5 dpc or later
because the extension of the migration area of melanoblasts in
16.5 dpc transgenic embryos was not observed in 14.5 dpc
embryos. In this stage, most of the melanoblasts were located in
the basal layer of the epidermis, where the SLF transgene was
expressed. As observed in the embryos of lethal spotting (ls/ls)
mutant mice (Yoshida et al., 1996), melanoblasts are able to
migrate through the epidermis of 14.5 dpc or later embryos.
Maintenance of c-Kit-independent stem cells of
melanocytes and their differentiation in the
postnatal skin of hk14-SLF transgenic mice
When SLF transgenic mice treated with anti-c-Kit functionblocking antibody ACK2 described in the previous section were
raised for a month (Fig. 6A, unshaved ventral view of the mice),
dorsal to lateral skin was uniformly repigmented (Fig. 6B) and
black pigmented spots appeared in ventral skin (Fig. 6C),
unhaired skin of ears, mouth, foot (Fig. 6A) and tail (Fig. 6D).
Considering that there was previously complete depigmentation
(Fig. 2) and no detectable TRP-2-expressing melanocyte
precursors (data not shown) in the skin of day 10 transgenic
mice treated with ACK2, the observed repigmentation seems to
be caused by the regeneration of melanocytes differentiated
from stem cells that were maintained independent of c-Kit
signaling. Each spot of Fig. 6C thus represents a single stem
cell clone. The pigmented spots of ventral skin continued to
expand and, 6 months after ACK2 administration, all spots were
completely fused (Fig. 6E). Histological analysis showed
uniformly distributed melanocytes in the epidermis of
repigmented skin (data not shown). This would further support
a role of SLF in the skin to promote migration of melanocytes
originated from small numbers of stem cells. The expansion of
pigmented spots is c-Kit dependent because the size of each
spot remained unchanged and the color of the spots faded after
the ACK2 treatment of 1-month-old SLF transgenic mice that
had developed clear pigmented spots due to the neonatal ACK2
treatment (compare Fig. 6G and H).
DISCUSSION
Multiple distinct developmental stages of melanocytes are
known to be affected by c-Kit and its cognate ligand SLF
(Yoshida et al., 1996a; Kluppel et al., 1997). By the continuous
expression of SLF in the epidermis, we have clearly found
evidence for the precise control of survival, growth,
differentiation and migration of melanocytes attained by SLFc-Kit signaling.
In normal mice, DOPA reaction-positive cells (melanoblasts
and melanocytes) found in the epidermis at birth decrease in
number from postnatal day 4 and then virtually disappear after
1 month except for those in hair follicles (Hirobe, 1984). The
number of these interfollicular melanocyte lineage cells was
increased in the skin of SLF transgenic mice after birth,
suggesting that the survival of melanocyte precursors was
extended by the locally produced SLF. Immediate loss of
TRP2-positive melanoblasts from the neonatal skin by the ckit function-blocking antibody (ACK2) injection and lack of
significant recovery of melanoblasts until day 10 after ceasing
injection of ACK2-treated mice (Fig. 2) also support the
conclusion that SLF promotes the survival of melanocyte
lineages in vivo. The fully pigmented melanocytes of SLF
transgenic mice were resistant to ACK2 treatment (H. Y. et al.,
unpublished data) suggesting that SLF affects the survival of
early stages of melanocyte lineage cells.
Unexpectedly, the ACK2-induced complete loss of
melanoblasts from the skin of SLF transgenic mice was
followed by the nascent regeneration of melanocytes a month
after ceasing the ACK2 treatment. This strongly suggests the
maintenance of c-Kit-independent stem cells for melanocytes
in the SLF transgenic skin. The presence of such a kind of cKit-independent melanoblasts was previously reported in the
hair follicles of normal mice (Nishikawa et al., 1991; Okura et
al., 1995). It was also shown that melanoblasts pass through
several alternating c-Kit-dependent and c-Kit-independent
stages during their normal development (Yoshida et al., 1993).
Thus, in the presence of ectopic SLF, c-Kit-independent
melanoblasts are likely to be constantly generated from the
more immature c-Kit-dependent melanoblasts. These c-Kitindependent cells are derived from c-Kit-dependent
melanoblasts because ACK2 treatment of SLF transgenic
embryos produced mice that were completely depigmented
even after a year (T. K. et al., unpublished data).
The existence of numerous melanoblasts in the skin of SLF
transgenic mice clearly suggests the promotion of proliferation
by the exogenous SLF. The blockage of further expansion of
the pigmented spots observed in the second ACK2 treatment
2920 T. Kunisada and others
of SLF transgenic mice (Fig. 6H) indicates that the
proliferation of the melanocyte lineage cells in the developed
interfollicular transgenic skin is promoted directly by the
exogenous SLF.
Differentiation of melanoblasts was also affected by SLF. In
SLFTg2-1, which showed extensive pigmentation of the entire
skin accompanied by massive mast cell accumulation
(Kunisada et al., 1998), pigmentation was visible in all regions
of newborn skin. In the case of one extraordinary transgenic
mouse, the whole epidermal region was severely pigmented at
birth with apparent epidermal hyperplasia possibly caused by
a massive number of mature melanocytes (data not shown).
Since melanoblasts/melanocytes found in the newborn skin of
normal mice are rarely pigmented, this may imply that SLF
promotes differentiation of melanocyte lineages. Interestingly,
an unpigmented ventral region is a characteristic of SLFTg2-
Fig. 4. Distribution of TRP2-positive melanoblasts in SLF
transgenic mice. Coronal sections through head were
stained by anti-TRP2 antibody. In 14.5 dpc embryo, TRP2positive melanoblasts were detected in the dermoepidermal
junction of oral epithelium cells (A). In this stage, TRP2positive cells (indicated by an arrowhead) were restricted to
the epithelium of alveolar ridges. While no TRP2-positive
cells were detected in the corresponding regions of
littermates (B), the basal cell layer of body wall skin
contained approximately equal densities of TRP2-positive
cells in SLF transgenic (C) and littermate (D) mice. In
newborn transgenic mice, the entire oral epithelium was
filled with TRP2-positive cells (E). In this stage, TRP2positive cells were detected in the primodium of teeth, as
indicated by an arrow in E. Body wall skin in this stage was
also filled with TRP2-positive cells in the transgenic mouse
(F). Note that the density of TRP2-positive cells was higher
in transgenic skin than in that of normal littermates (H). No
TRP2-positive cells were found in the oral epithelium of
normal littermates (G). In the ears of 2-month-old normal
mice, pigmented melanocytes (arrowhead) were mostly
distributed in the dermal area (J) whereas melanocytes
(arrowhead) were restricted to the epidermis (I). Asterisks
in I and J indicate hair follicles. Abbreviations: oc, oral
cavity; tg, tongue; hf developing hair follicle; pmt,
primodium of molar tooth; nc, nasal cavity. Scale bar
represents 100 µm in A-H and 50 µm in I and J.
1 (shown by an arrow in Fig. 1H). The relatively abundant
transgenic SLF may allow migrating melanocytes to
differentiate into mature melanocytes before reaching
abdominal skin. This observation thus provides additional
support that SLF may act as a stimulus for melanocyte
differentiation in vivo. While it is well established that in vitro
culture of neural crest cells in the presence of SLF promotes
mainly the survival of melanoblasts (Murphy et al., 1992; Steel
et al., 1992; Morrison-Graham and Weston, 1993), SLF or
other extracellular signals have been reported to activate the
transcription factor MITF (Hemesath et al., 1998; Bertolotto et
al., 1998) in melanocyte cell line. As MITF is known to have
the capability to differentiate fibroblasts into melanocyte-like
cells (Tachibana et al., 1996), SLF may promote the
differentiation of melanocyte lineages via MITF in the skin of
the transgenic mice.
Development of melanocytes and steel factor 2921
Fig. 5. Coat phenotypes of W57/W57 and W/W57 mutant mice (A) or
bt/bt mutant mouse (B) crossed with SLF transgenic mice.
Phenotypes were consistent in the several independent crosses of
both mutants.
Promotion of the migrative/chemotactic activity by SLF
was suggested previously in melanocyte, mast cell and germ
cell lineages during embryonic development (Galli et al.,
1993). One direct experimental approach taken has been in
vitro measurement of chemotactic activity of c-Kit-expressing
endothelial cells (Blume-Jensen et al., 1991) or mast cells
(Meininger et al., 1992) in response to recombinant SLF.
Human melanocytes cultured in vitro did not show the
induction of chemotactic activity by SLF (Horikawa et al.,
1995). Although subcutaneous injection of SLF resulted in the
appearance of a large number of mast cells at the injection site
in the mouse (Zsebo et al., 1990; Tsai et al., 1991) and
melanocytes in humans (Harrist et al., 1995; Costa et al.,
1996), the circulating nature of mast cell precursors and the
presence of melanocytes before SLF injection in the human
skin also raise the possibility that SLF might only have
promoted growth and differentiation, not migration of these
cells. By using a cytokeratin promoter, we have induced
targeted expression of SLF in the whole epidermal layer
including the regions where no pigmentation is observed in
the normal mouse. In these regions, especially in the oral
epithelium and footpads, no melanocyte precursors are
detected in any developmental stages of normal mice.
Accordingly, melanoblasts found in these regions of SLF
transgenic mice are likely to have had their limit of migration
extended by the exogenous SLF. Simple overgrowth might not
explain the observation that the density of melanoblasts in
Fig. 6. Repigmentation of SLF transgenic mice treated with ACK2.
SLFTg1-1 mice were injected with ACK2 on days 0, 2 and 4 after
birth and raised for a month. Most of the hair is unpigmented in the
belly skin (A) and a significant portion of the hair of the back skin is
pigmented (B). The interfollicular region of the back skin is
completely pigmented (shaved area of B) and pigmented spots are
observed in the belly skin (C), tail (D), foot and external genitalia
(A). When these ACK2-treated mice was raised for 2 additional
months, the belly skin (E) and tail (F) were completely pigmented.
This expansion of pigmented spots was prevented by a second
administration of ACK2 antibody; the 1-month-old mouse shown in
G was injected with ACK2 every other day for a total of 3 injections
and photographed after a month (H).
transgenic embryos remained comparable to that of normal
littermates when precursors had reached the oral epithelium at
16.5 dpc (Fig. 4C,D). Likewise, in Slcon mutant mice,
melanocytes are known to accumulate in the reproductive tract
and oviduct and in these tissues ectopic expression of SLF
mRNA was observed (Bedell et al., 1995). The pigmentation
defects, i.e. the absence of melanocytes in the mid-trunk
region of W/W57, W57/W57 or bt/bt mutations were
significantly rescued by crossing with SLF transgenic mice.
Under the transgenic background, as shown in Fig. 5,
pigmented areas of the skin were enlarged (W/W57 ; tg/+ and
bt/bt; tg/+) or newly formed on the originally white coat
(W/W57; tg/+). Apparently, even in a white coat phenotype
such as W/W57, melanoblasts generated as early as 9.5 dpc
survive until 12.5 to 13.5 dpc, when they enter the basal layer
of epidermis and utilize transgenic SLF expressed in situ.
Provided that there exist other factor(s) independently
conferring migratory activity on melanoblasts, these surviving
melanoblasts at least migrate normally and distribute over the
whole coat skin. Subsequently, more or less randomly
2922 T. Kunisada and others
distributed pigmented patches derived from these
melanoblasts might be produced. However, in our
observations, pigmented areas appear exclusively in the head
and posterior trunk regions as shown in Fig. 5. This
distribution pattern is consistent with the observation that
TRP2-positive melanoblasts were far more abundant in these
regions than in the mid-trunk region (Yoshida et al., 1996).
Therefore, surviving melanoblasts in these W and Sl mutants
do not seem to actively migrate during 9.5 to 12.5 dpc.
However, the possibility that the main function of the SLF
transgene in these genetic cross experiments is only to
complement growth and/or survival of affected melanoblasts
of the W or Sl mutant can not be ruled out. Melanoblasts
derived from the most recently generated neural crest cells
might be saved by transgenic SLF after the entry into
epidermis. These saved melanoblasts might generate
pigmented areas in the head and tail regions. Another line of
evidence came from the pigmented spots formed after ACK2
treatment of SLF transgenic mice. They first appeared as small
dots and then rapidly increased their size, and this process was
blocked by the second ACK2 treatment. Taken together, these
results strongly suggest a migratory role of SLF; however, the
phenotype expressed as an alteration of coat pigmentation
pattern could not be rigorously attributed to either proliferative
or migratory roles of SLF. The additional observation that
melanocytes in the ear of SLF transgenic mouse were
distributed closely under the epidermal layer whereas they
mostly reside in the dermal layer in the normal control might
indicate the migratory or chemoattractive roles of SLF on the
melanocyte lineage cells.
The various pigmentation phenotypes that appeared
among SLF transgenic mice indicate that the expression level
of SLF affects the distribution of melanocytes in the skin.
Expression levels of transgenic SLF in the skin examined are
SLFTg2-1>SLFTg1-1>SLFTg4-3 (Kunisada et al., 1998; T.
K., unpublished data). In SLFTg4-1 to SLFTg4-3, tail,
external genitalia or ears were pigmented (Fig. 1I-K). In
SLFTg1-1 and SLFTg1-2, unhaired skin as well as the entire
haired skin were pigmented (Fig. 1D,E). As described in the
previous paper, the entire skin of SLFTg2-1, SLFTg2-2,
SLFTg2-3 and SLFTg2-4 mice was severely pigmented and
varying sizes of completely unpigmented areas appeared in
the belly, feet or tail. Although they are not found in haired
skin, mature melanocytes reside in tail, external genitalia and
ears of normal mice; therefore, a small amount of SLF may
promote the survival of melanoblasts in these tissues of
SLFTg4 mice. Along with the reduction of endogenous SLF
in the haired skin region, surviving melanoblasts in the
haired skin may disappear. For the continuous proliferation
or differentiation of melanocytes in the haired skin, the
expression of SLF at greater levels than that of SLFTg1 skin
might be required.
In summary, analysis of the SLF transgenic mice described
in this report has clearly demonstrated that SLF acts as a key
factor to support the temporally and spatially coordinated
survival, proliferation, differentiation and migration of
melanocyte lineages. In addition, some of the transgenic lines
established enabled us to study these developmental processes
in the skin of postnatal mice. In particular, the repigmentation
process after ACK2 treatment of SLF transgenic mice reported
here is similar to the repigmentation phase of vitiligo patients.
The use of the transgenic mice may help to search for an
effective method to promote vitiligo repigmentation.
We thank Dr. T. Shibahara of the Laboratory Animal Research
Center of Tottori University for maintenance of the transgenic mice
and Ms. T. Shinohara for secretarial assistance and also thank Dr E.
Fuchs for hk14 promoter sequense and Dr. V. Hearing for anti-TRP2 antibody. This work was supported in part by grants from the
Ministry of Education, Science, Sports and Culture of Japan, Special
Coordination Funds for Promoting Science and Technology from the
Science and Technology Agency of Japan, Uehara Memorial
Foundation (Tokyo, Japan). We also thank for the support from the
Cellular Technology Institute, Otsuka Pharmaceutical Co., Ltd
(Tokushima, Japan).
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