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A possible mechanism responsible for the melanocyte distribution of the perineal pigment spot of the mouse.

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A Possible Mechanism Responsible for the Melanocyte
Distribution of the Perineal Piament
Spot of the Mouse'
II
JOHN H. SCHULTZ2 AND HERMAN B. CHASE
Department of Biology, Brown University,
Providence, Rhode lsland
ABSTRACT
The perineal pigment spot of the C57 Black mouse was examined
macro- and microscopically in animals ranging in age from 14 days post-fertilization
to 28 days post-parturition. Dendritic pigment-containing cells first become evident i n
the dermis of the area on the sixteenth day post-fertilization. Throughout later stages
of development, these pigmented cells continue to be restricted to the dermis of this
area. Since the dopa treatment reveals the presence of a melanin synthesizing system
within these cells, they are true melanocytes which have become restricted to this
layer of the integument.
Histological examination reveals a population of melanocytes in the epidermis
and/or hair follicles also. These are rarely found over the center of the area but are
seen in increasing numbers laterally from the ventral midline. In contrast, dermal
melanocytes are heavily concentrated near the ventral midline but decrease i n numbers
laterally. There are, therefore, two opposing but complementary gradients of melanocytes in the integument of the pigment spot. This suggests the possibility that a precocious differentiation of the basement membrane in this area during embryonic development inhibits the passage of melanoblasts into the epidermis thereby causing
them to be restricted to the dermis.
The neural crest origin of presumptive Review, Markert and Silvers, '56). While
pigment cells of the mammalian integu- the widespread distribution of melanocytes
ment was firmly established by the defini- in extra-integumental tissues of other vertetive experimental work of Rawles ('47, '53). brates is well known, recent work by
She was able to demonstrate that those Nichols and Reams ('60) and Mayer and
cells capable of ultimate differentiation in- Reams ('62) confirms previous evidence of
to melanocytes come from tissues contain- a similar distribution in mammals. In their
ing presumptive neural crest, histologically special PET strain of mice they have disrecognizable neural crest, or from cells covered a population of melanocytes dismigrating from the neural crest. Although tributed throughout organs and tissues
the origin of integumental melanocytes is where pigmented cells had not been demthus well established, the precise mecha- onstrated previously in other strains. In
nism by which these cells reach their final fact, melanocytes were found to be ubiquilocation in the skin has not yet been deter- tous in their distribution and were absent
mined although various schemes have been only from the connective tissue of the gut
mucosa.
proposed.
Although the epidermis and hair norIn studies of the later stages of skin pigmentation in mammals, the attention of mally contain most of the pigment-producmost investigators has been focused pri- ing cells of the integument, there are some
marily on the study of epidermal pigment areas in the mouse in which pigmented,
(Masson, '48; Billingham and Medawar, dendritic cells are found in large numbers
'53; Holmes, '53; Reynolds, '54) and the in the dermis. These areas include the
pigment of hair (Billingham and Medawar, muzzle, ears, soles of the feet, genital
'50; Chase, Rauch, and Smith, '51; Chase, papilla, scrotum, and tail (Steiner-Wour'58). Other studies have shown that pig- lisch, '25; Holmes, '53; Reynolds, '54;
ment cells are also present in the men1 Supported by U.S.P.H.S. grants C-592 and 2G-329
University.
inges, the harderian and thymus glands, to 2Brown
Predoctpral Trainee in Genetics (U.S.P.H.S. Trainand the choroid and retina of the eye (cf. ing Grant in Genetlcs 26-329).
59
60
JOHN H. SCHULTZ AND HERMAN B. CHASE
Markert and Silvers, '56; Billingham and
Silvers, '60). With the exception of the
early work of Steiner-Wourlisch, most of
the more recent studies of dermal pigment
have been incidental to studies of epidermal pigment or hair. On the assumption
that the neural crest is the origin of potential pigment cells and that their migration
takes place between the ectoderm and
mesoderm during embryonic development,
the examination of an area containing pigment cells in both the dermis and the
epidermis could possibly help to explain
some of the developmental mechanisms
which lead to the restriction of the potential melanocytes to either one or the other
or both of the tissue layers. This paper is
a report of such an undertaking.
The heavily pigmented perineum of the
C57 Black mouse, the so-called male pigment spot, and the homologous area of the
female, were the regions selected for study.
In darkly pigmented strains of mice this
area is intensely pigmented in the neonatal
male and is often used as an aid when
sexing the animals at birth. It is situated
between the genital papilla and the anus
and later becomes the definitive scrota1 sac
of the male. This particular region was
selected because it has a large amount of
pigment confined largely to the dermis, it
is readily accessible, and i t is amenable to
experimentation.
containing the pigment was examined under the dissecting microscope.
For general histological procedures,
whole fetuses up to sixteen days post-fertilization were fixed in Bouin's fixative.
Since the keratin of the skin of later fetuses and postnatal animals interferes with
the penetration of the fixative, only the pigmented area was removed and fixed. After
fixation and paraffin embedding, the tissues were sectioned at 6 p, stained lightly
with Harris' hematoxylin and mounted under balsam.
In order to determine if the pigmentcontaining cells were the same cells which
had produced the pigment, a parallel series
of tissues was taken and subjected to the
dopa paraffin technique of Becker ('48).
Following the dopa treatment these tissues
were also sectioned at 6 I-I and stained
lightly with Harris' hematoxylin.
To facilitate comparison between control
and treated tissues, another series of postnatal tissues was selected. The tissue samples were cut in half longitudinally; one
half was incubated in the buffered dopa
medium while the control half was incubated in the same medium but without the
dopa. The two halves from each piece
were reunited into a single block of paraffin, cut, stained, and mounted as previously described. Using this method, comparisons between treated and control sections could be made very rapidly.
MATERIALS A N D METHODS
All mice used in the current study were
from the highly inbred C57 Black/lOCh
strain maintained at Brown University.
Groups of mature animals were mated and
the embryos were removed 12 or more days
after the initial matings. The criteria for
determining fetal age (Griineberg, '43)
were then applied so that the developmental stages were standardized and the
variability between litters was minimized.
Animals ranging in age from 14 days of
gestation to 28 days post parturition were
examined grossly and histologically. For
gross examination fetuses were removed
and placed in a 0.9% saline solution. The
area of the potential pigment spot was
then examined under a dissecting microscope (up to X 30). Neonatal and postnatal animals were sacrificed by cervical
dislocation or etherization and the tissue
RESULTS
Fetuses examined macroscopically prior
to the eighteenth day of development show
no obvious pigmentation within the area
of the pigment spot. The deeply folded
epidermis, however, would tend to obscure
any pigment which might be present. Histological examination, on the other hand,
reveals a few scattered, highly dendritic
dermal pigment cells on the sixteenth day
(fig. 1 ) . These cells exhibit a weak reaction to the dopa treatment at this time.
For the next three days there is a rapid increase in the number of these cells along
with a stronger reaction to the dopa treatment. By the day of birth, which in this
strain is usually the nineteenth or twentieth day, the outline of the pigment spot can
be seen readily with the naked eye. Upon
first appearance, the melanocytes of the
PIGMENT SPOT MELANOCYTES
fetal dermis are highly dendritic with randomly oriented processes; at birth, however, the dendrites are arranged mostly
parallel to the surface of the skin. Cross
sections through the area show marked
differences in melanocyte distribution between the two sexes, males having a heavy
concentration of pigment cells in the
dermis of the pigment spot in contrast to a
lesser concentration in the homologous
area of the female (figs. 2, 3 ) . Also, the
pigment cells of the males appear to have
longer dendrites which are more heavily
pigmented.
Common to both at all stages studied is
a median raphe at the ventral midline.
This is indicated by an indented thickened
epidermis below which is an increased concentration of dermal fibroblasts. In this
zone of thickened connective tissue few or
no pigment cells are found. Cross sections
through the spot disclose heavy concentrations of dermal melanocytes close to this
ventral midline but slightly lateral to it.
These concentrations diminish gradually
toward the margins of the spot until dermal melanocytes disappear or are no longer
evident at the edges. In the basal layer of
the epidermis and in many of the developing hair follicles at the edges of the area,
a few pigmented, dendritic cells can also
be seen. Medially, however, fewer and
fewer epidermal pigment cells are evident
and they are seldom found over the midline
of the area.
Two different zones can be distinguished
within the reticular layer of the dermis in
the area. The zone closest to the epidermis
has very closely packed cells whereas that
of the deeper layer is characterized by a
much looser arrangement of connective tissue cells. These layers also differ in their
melanocyte types. Melanocytes in the upper dense layer have heavily pigmented
perikarya and long filamentous processes
ramifying in all directions. Cells of this
type differ from those found in the deeper,
more loosely packed layer, the latter being
bipolar, less heavily pigmented, and arranged parallel to the long axes of the deep
connective tissue cells (figs. 6, 7).
Along with an increase in pigment within the melanocytes, the most striking
change is in the orientation of the dendrites during the first few days following
61
birth. Whereas at birth, the processes
were arranged mostly parallel to the skin
surface, they now begin to ramify in all
directions and exhibit an increased overlapping of each other. Pigment cells in the
deep layer on the other hand, retain their
bipolar character and remain oriented parallel to the connective tissue cells.
By the third day, the dopa reaction has
reached maximum intensity in the epidermal, dermal and hair bulb melanocytes
(fig. 4). The hair follicles begin to penetrate deeper into the upper layer of the
dermis and in so doing, they force the dermal melanocytes aside adding to the disorientation of the dendrites. Because of
the greater density of pigment cells in
males, this change from the original arrangement is more apparent in males than
in females. Although this downgrowth results in a very close association between
the follicles and the dermal melanocytes,
these pigment cells are not incorporated
into the follicles.
The bulbs of the follicles begin to penetrate the deep dermis by the fifth day and
the dermal melanocytes in the upper layer
now occupy the spaces between them. At
this point in development, the perikarya of
the individual pigment cells are not usually
found close to the dermal-epidermal junction, but instead, are usually at or below
the midpoint of the follicles. The dendrites
ramify in all directions but appear to be
oriented toward the skin surface in many
cases (fig. 6 ) . Toward the lateral edges
of the pigment spot, melanocytes of the
upper layer of the dermis decrease in
numbers while those of the deep dermis
disappear altogether. Of the hair follicles
present, the number of those containing
pigment decreases medially. Although
some pigment cells are seen in the basal
layer of the epidermis, most are confined
to or are closely associated with developing hair follicles.
Tissues taken from neonatal and postnatal animals prior to the seventh day of
development show two distinct gradients of
pigment cells. Dermal pigment cells are
heavily concentrated near the ventral midline but diminish in numbers laterally and
gradually disappear at the margins of the
pigmented area. In contrast, pigment cells
are absent from the epidermis over the ven-
62
JOHN H. SCHULTZ AND HERMAN B. CHASE
tral midline but appear gradually in increasing numbers laterally. This impression of two opposing gradients is most obvious in the dopa treated sections (fig. 4).
Beginning on the seventh day there is a
major change in the distribution of melanocytes. After this time they are no longer
found in the epidermis. Whereas the pigment cells of the dermis persist in their
original location, those originally seen in
the epidermis now appear to be confined
to the hair bulbs where the pigment granules are incorporated into the actively
growing hairs (fig. 8 ) .
The intense dopa reaction continues in
both the dermal and hair follicle melanocytes at this time. Also, there is a slight,
but nevertheless apparent, difference in
the intensity of pigmentation in the dermal
melanocytes seen in untreated sections.
Those close to the surface are very heavily
pigmented whereas those closer to the level
of the hair bulbs are less heavily pigmented (fig. 7).
The tips of the developing hairs first
emerge through the surface of the skin
around the periphery of the spot on the
eighth day. For the next two days there is
a gradual emergence of hairs which progresses medially, and by the tenth day the
entire area is covered with actively growing hairs which have broken through the
skin surface. Near the edges of the spot,
there is an increase in the number of pigmented hairs. Typical pelage hairs are intermingled with pigmentless hairlets and
special hairs. In fact, most of the hair
bulbs found close to the center of the spot
are non-pigmented.
The relationship between the dermal and
epidermal pigment granules remains essentially unchanged up to the fifteenth day at
which time a few scattered club hairs can
be seen near the periphery of the spot.
For the next three days increasing numbers of club hairs appear in the area and
by the eighteenth day all of the follicles
have entered the resting stage. Ten days
later, some of the follicles enter the anagen
stage and begin a second hair growth cycle.
During the quiescent phase of the hair
growth cycle, melanocytes cannot be demonstrated in the hair bulbs either before or
after dopa treatment.
Although the ultimate fate of the dermal
pigment cell granules is undetermined at
this time, they do not appear to be passed
on to neighboring cells as is the case with
hair and epidermal pigment. Single granules which were observed at various levels
within the dermis, may be either intra- or
extracellular. The occasional clumps of
pigment which appear not to be associated
with a particular pigment cell, could possibly be the pigmented ends of dendritic
processes which have become pinched off
from the main cell body.
Dendritic, melanin-containing cells are
found in both layers of the dermis in all
stages studied, and the concentration and
arrangement of reactive cells after dopa
treatment is the same as that which is
found in the control sections, with few exceptions. Hair follicles in the process of
growth contain large numbers of dopa-reactive cells, but during the early stages these
are confined primarily to the external
sheath which is continuous with the basal
layer of the epidermis. In fact, the dopapositive melanocytes of the basal layer are
almost always closely associated with the
developing hair follicles and are seldom observed without this association. Females
show more reactive cells in the basal layer
than do males but at the same time show
fewer in the dermis. It is also of interest
that females have a greater number of pigmented hairs of all types in the homologous area than do males.
DISCUSSION
It has been suggested that pigment-containing cells of the dermis are phagocytes
which have ingested pigment granules produced by epidermal melanocytes (Becker,
'27; Masson, '48; Holmes, ' 5 3 ) . Although
this phagocytic possibility may exist in
some tissues, it is not the mechanism at
work within the pigment spot, because the
dopa reaction shows that the dermal cells
which contain this pigment also have the
active enzyme system necessary for pigment production. Since this enzyme system is contained on or within the pigment
granules, a rapid ingestion of such granules by phagocytosis is not impossible, but
the low incidence of epidermal melanocytes over the area makes such a mechanism highly improbable. On the basis of
PIGMENT SPOT MELANOCYTES
these observations, it may be concluded
that these dermal pigment-containing cells
are true melanocytes which have become
restricted to the dermis during embryonic
development.
The inductive influences of one tissue on
another are well documented in the biological literature. Rawles ('47) and Nichols
and Reams ('60 j have suggested that there
is an agent, possibly humoral which exerts
an influence on the melanocytes of the
mouse. A suggestion of an interaction of
some type is based on the observation that
melanocytes in the lower level of the dermis are not as heavily pigmented nor as
highly branched as are those closer to the
skin surface. There may be factors, however, intrinsic to the tissue environment in
the different tissue layers which could also
account for these differences of expression.
Based primarily on the work on Rawles
('47, '53 j , the neural crest has been firmly
established as the ultimate source of potential melanocytes in mammals. During embryonic development, these potential melanocytes migrate ventrally between the
potential epidermis and the mesenchyme,
normally filling the area to the ventral midline. By far, the preponderance of these
cells become incorporated into the epidermis where they are ultimately found as
follicular pigment cells after hair follicles
have developed by proliferation of the basal
layer of the epidermis (Chase, Rauch, and
Smith, '51). In the case of the pigment
spot, this arrangement of pigment cells is
reversed. A greater number of melanocytes
is found instead in the dermis with very
few in the epidermis. This heavy concentration of melanocytes in this area could
be due to an overgrowth of the mesenchyma1 tissues as they meet at the ventral midline. It is also possible that the presumptive epidermis is slower in development at
the same time in this region. Also, the
mechanism could be in the reverse order.
Since little is known of the embryonic development of this tissue, any of these
mechanisms could explain the heavy concentration of dermal melanocytes. Mayer
and Reams ('62) have suggested an inturning or spilling over to account for the
melanocyte distribution in the leg musculature of their PET mice. In the pigment
spot the failure of the melanocytes to enter
63
the epidermis could be the result of a barrier, possibly the basement membrane,
which probably forms before the melanoblasts can migrate into the epidermis, thus
preventing this layer from receiving its
"normal" complement of pigment cells.
Evidence to support such an idea comes
from the recognition of two opposing pigment gradients, one associated with the
dermis, and a complementary one associated with the epidermis. In the development of the integument in other areas of
the body surface, there must be a relationship between the potential dermis and epidermis which allows the migration of
melanoblasts into the epidermis before the
membrane forms. It appears, therefore,
that the normal timing sequence of basement membrane formation is precocious in
the area of the pigment spot, possibly the
result of differing rates of growth of the
two layers of the integument. This precocious development of the membrane could
inhibit the passage of melanoblasts into the
epidermis. Schumann ('60) suggests a
somewhat similar mechanism in the analysis of the white blaze of the mouse in
which there are postulated to be genetic
factors at work which cause a delayed migration of melanoblasts into the region.
This causes the melanoblasts to arrive at
the site of the white blaze after the epidermis has become impervious to their
entrance (cf. Chase, '39, for white spotting
in the guinea pig). This idea of differential migration is in contrast to that of
Markert and Silvers ('56 j who have suggested that areas devoid of pigment are
not the result of differential migration, but
rather are due to factors within the tissue
environment. Although the effect of tissue
environment cannot be ruled out, it seems
more likely that differential growth and
concomitant differential migration are the
factors responsible for the melanocyte arrangement in the pigment spot.
LITERATURE CITED
Becker, S. W. 1927 A systematic study of the
pigment of human skin and the upper mucous
membranes with special consideration of pigmented dendritic cells. Arch. Derm. Syph.,
N. Y.,16: 259-290.
1948 Dermatological investigations of
melanin pigmentation. In: The Biology of
Melanomas. ( R . W. Miner, ed.). Special
Publ., N. Y. Acad. Sci., 4: 82-125.
64
J O H N H. S C H U L T Z A N D H E R M A N B. C H A S E
Billingham, R. E., and P. B. Medawar 1950
Pigment spread in mammalian skin: Serial
propagation and immunity reactions. Heredity,
4: 141-164.
1953 A study of the branched cells of
the mammalian epidermis with special reference to the fate of their division products.
Phil. Trans., 237: 151-171.
Billingham, R. E., and W. K. Silvers 1960 The
melanocytes of mammals. Quart. Rev. Biol.,
35: 1 4 0 .
Chase, H. B. 1939 Studies on the tricolor pattern of the guinea pig. 11. The distribution of
black and yellow as effected by white spotting
and by imperfect dominance in the tortoise
shell series of alleles. Genetics, 24: 622-643.
1958 The behavior of pigment cells and
epithelial cells in the hair follicles. I n : The
Biology of Hair Growth. (W. Montagna and R.
E. Ellis, eds.). Academic Press, New York, pp.
229-237.
Chase, H. B., H. Rauch and V. W. Smith 1951
Critical stages of hair development and pigmentation in the mouse. Physiol. Zool., 24:
1-10.
Griineberg, H. 1943 The development of some
external features in mouse embryos. J. Hered.,
34: 89-92.
Holmes, R. L. 1953 Patterns of cutaneous pigmentation: Rodents. J. Anat. Lond., 87: 163168.
Markert, C. L., and W. K. Silvers 1956 The effects of genotype and cell environment on melanoblast differentiation in the house mouse.
Genetics, 41 : 4 2 9 4 5 0 .
Masson, P. 1948 Pigment cells in man. In:
Biology of Melanomas. (R. W. Miner, ed.). Special Publ., N. Y. Acad. Sci., 4: 15-51.
Mayer, T. C., and W. M. Reams, Jr. 1962 A n
experimental analysis and description of the
melanocytes i n the leg musculature of the PET
strain of mice. Anat. Rec., 142: 4 3 1 4 4 1 .
Nichols, S. E., and W. M. Reams, Jr. 1960 The
occurrence and morphogenesis of melanocytes
in the connective tissue of the PET/MCV mouse
strain. J. Embryol. Exp. Morph., 8: 24-32.
Rawles, M. E. 1947 Origin of pigment cells
from neural crest in the mouse embryo. Physiol. Zool., 20: 248-266.
1953 Origin of the mammalian pigment cell and its role in the pigmentation of
hair. In: Pigment Cell Growth. (M. Gordon,
ed.). Academic press, New York, pp. 1-13.
Reynolds, J. 1954 The epidermal melanocytes
of mice. J. Anat. Lond., 88: 45-60.
Schumann, H. 1960 Die Entstehung der Schekung bei Mauser mit weiser Blesse. Dev. Biol.,
2: 501-514.
Steiner-Wourlisch, A. 1925 Das melanotische
Pigment der Haut bei der grauen Hausmaus
(Mus musculus). Z . Zellforsch. u. mikr. Anat.,
2: 453-479.
PLATE 1
EXPLANATION OF FIGURES
Dendritic pigment cells of the fetal dermis, first seen at day 16 postfertilization. Harris’ hematoxylin. x 950.
Cross section through the pigment spot of a neonatal male. The concentration of dermal melanocytes is heavy near the midline, on the
left i n the photograph, but diminishes laterally, toward the right.
Harris’ hematoxylin. x 95.
Cross section through the homologous area of a neonatal female. The
concentration of dermal melanocytes is much less than that seen in
a male of the same age. Harris’ hematoxylin. x 95.
Dopa treated cross section through the spot of a three day old male.
A heavy concentration of reactive cells can be seen i n the dermis near
the center of the spot, on the right. A few are also visible within
the basal layer of the epidermis, toward the left. None, however, are
visible within the basal layer over the center of the spot. Dopa and
Harris’ hematoxylin. X 95.
Control section to that seen i n figure 4. Melanocytes of the epidermis
are not visible. Harris’ hematoxylin. X 95.
Sagittal section through the spot of a five day old male. The dermal
melanocytes close to the skin surface are highly branched and heavily
pigmented. Those of the lower layer are less heavily pigmented.
Harris’ hematoxylin. X 95.
Sagittal section through the pigment spot of a seven day old male.
Dermal pigment cells are concentrated around the hair follicles but
are not a n integral part of them. Those of the deeper connective
tissue layer are bipolar and arranged parallel to the long axes of the
connective tissue cells. Harris’ hematoxylin. x 95.
Dopa treated cross section of the pigment spot of a ten day old male.
The dermal pigment gradient increases toward the center, on the
right. Follicular pigment increases in the opposite direction. Dopa
and Harris’ hematoxylin. X 48.
PIGMENT SPOT MELANOCYTES
John H. Schultz and Herman B. Chase
PLATE 1
65
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