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Copyright © 1998 by the Genetics Society of America

The Murine Misty Mutation: Phenotypic Effects on Melanocytes,


Platelets and Brown Fat

Elena V. Sviderskaya,* Edward K. Novak,† Richard T. Swank† and Dorothy C. Bennett*


*Department of Anatomy and Developmental Biology, St. George’s Hospital Medical School, London SW17 0RE, and †Molecular and
Cellular Biology Department, Roswell Park Cancer Institute, Buffalo, New York 14263
Manuscript received June 6, 1997
Accepted for publication September 15, 1997

ABSTRACT
Although the recessive murine mutation misty (m) is well known, its phenotype has never been reported
beyond brief descriptions of a dilution of coat color and white spotting of the belly and extremities, sug-
gesting a developmental mutation. A report in abstract has also suggested effects on white fat and body
weight. Here, we report effects of the homozygous misty mutation on an unusual combination of three cell
types: melanocytes, platelets, and brown fat. Brown fat appeared to be completely absent from all expected
locations in neonatal m/m mice. A prolonged bleeding time was observed; platelet count and platelet sero-
tonin and ATP levels were normal, but the level of ADP in m/m platelets was low. Primary cultures and im-
mortal lines of melanocytes from m/m mice showed several abnormalities. There was a marked deficiency
in net proliferation, suggesting that the color dilution and spotting in vivo may result from reduced num-
bers of melanocytes and their precursors. m/m melanocytes were also hyperdendritic in morphology, over-
produced melanin, and had deficient responses to the cAMP agonists cholera toxin and melanocyte-stimu-
lating hormone, which normally promote melanin production. The misty gene product may be involved in
adenine nucleotide metabolism or signaling.

M ELANOCYTES are ideally suited to genetic anal-


ysis, because germline mutations affecting pig-
mentation are often nonlethal, readily visible and thus
tokine endothelin 3 and its receptor Ednrb, and the
transcription factors Pax-3 and Mi/MITF (Bennett
1993; Baynash et al. 1994; Jackson 1994). Most of
easily selected. About 85 coat-color loci are now known these genes have also proved to be loci for human ge-
in the mouse [Silvers 1979; Bennett 1993; Doolit- netic defects, including piebald trait (W/KIT ), Hirschs-
tle et al. 1996; Mouse Genome Database (MGD), June prung’s disease (s/EDNRB) and Waardenburg’s syn-
1997. Mouse Genome Informatics, The Jackson Labo- drome types 1 (Sp/PAX3) and 2 (mi/MITF ) (Bennett
ratory, Bar Harbor, ME, http://www.informatics. 1993; Baynash et al. 1994; Tassabehji et al. 1994).
jax.org/]. Many of these are involved in the function of Another such spotting locus is misty (m). Although it
melanocytes, but around 33 act in their development is well known, as an anchor locus in chromosomal map-
(Bennett 1993; Mouse Genome Database); these are ping, little is understood about this mutation. It arose
the spotting loci, which reduce the area of skin and spontaneously in the DBA/J strain of mice over 50
hair that is pigmented, while not usually affecting eye years ago (Woolley 1941, 1945). It is located on chro-
color (Silvers 1979). The technique of melanocyte cul- mosome 4, 7.2 cM distal to brown. There is no molecu-
ture, pioneered with human (Eisinger and Marko lar information on this locus, although several PCR-
1982) and mouse (Sato et al. 1985; Bennett et al. 1987; based sequence markers have been mapped near to
Tamura et al. 1987) cells, has proved valuable in molec- misty (Fiedorek and Kay 1994), and a recent abstract
ular studies of color genes, for example, the albino and has suggested a deficiency in body weight and white fat
brown loci, encoding the melanosomal enzymes tyrosi- in older m/m mice (Truett et al. 1996). Homozygous
nase and TRP1 (Yamamoto et al. 1989; Bennett et al. misty mice typically show paler pigmentation on black,
1990; Jackson and Bennett 1990). The sites of a num- brown and agouti backgrounds, interpreted as a dilu-
ber of the developmental (spotting) mutations have tion of eumelanin (black melanin), but also have a
been cloned. These include the loci Sl, W, ls. s. Sp and white tail-tip and feet and a belly streak or spot, suggest-
mi, encoding, respectively, the growth factor SCF (stem ing reduced numbers of melanocytes or their later pre-
cell factor) and its tyrosine kinase receptor kit, the cy- cursors (Silvers 1979). As no illustration of these coat
features has been published to our knowledge, we in-
clude one here (Figure 1).
Corresponding author: Dorothy C. Bennett, Department of Anat- We became interested in misty as a candidate devel-
omy and Developmental Biology, St. George’s Hospital Medical
School, Cranmer Terrace, London SW17 0RE, UK. opmental mutation that might affect late melanoblast
E-mail: dbennett@sghms.ac.uk development. Because so little is known of the misty

Genetics 148: 381–390 (January, 1998)


382 E. V. Sviderskaya et al.

phenotype, we initially performed a general histologi- Histology: Mice homozygous for the misty mutation but on
cal survey of neonatal misty mice to check for gross ab- an outbred background (C3H/HeH 3 101/H) carrying vari-
able agouti (A, ae) and brown (B, b) alleles were obtained from
normalities other than in color. As another general
C. Beechey (MRC Radiobiology Unit, Didcot, UK). misty was
study, we tested whether misty formed one of the group separated from the other mutations and combined with non-
of 14 pigmentary mutations, such as beige, ruby-eye and agouti (black: a/a, B/B), a standard genetic background for
pale ear (Swank et al. 1991; Swank et al. 1996; Bennett study of color mutations, by backcrossing to the C57BL/6J
1993), that also produce platelet storage pool defi- strain every two generations, alternated with sibling mating
and selection for the misty nonagouti phenotype. After about
ciency (Rao 1990) and, generally, lysosomal abnormali-
eight backcross generations, when no other color alleles were
ties as well. Similar disorders occur in humans (Spritz being segregated, a/a, m/m litters were obtained from parents
and Hearing 1994; King et al. 1995). For example, of this genotype. One-day-old mice were killed and fixed
Chediak-Higashi syndrome is homologous to the mu- whole in formal-calcium (formalin with calcium acetate).
rine beige mutation (Barbosa et al. 1996; Nagle et al. They were paraffin embedded, and transverse 7 mm sections
were cut at various body levels. These were dewaxed, stained
1996; Perou et al. 1996), while pale ear was recently
with Ehrlich’s alum hematoxylin and eosin, and mounted in
reported to be a homolog of Hermansky-Pudlak syn- Histomount for bright-field microscopy and photography.
drome (Feng et al. 1997). Lastly, we prepared cultures Feeder cells: Keratinocyte feeder cells were from the im-
of m/m melanoblasts and melanocytes to test for any ef- mortal XB2 murine keratinocyte line, provided by Jim Rhein-
fect on cell proliferation, differentiation, survival or wald (Rheinwald and Green 1975), and were prepared as
described previously (Bennett et al. 1989), using mitomycin C
other property of this lineage, with striking results.
treatment for 3 hr followed by resuspension and freezing.
Feeder cells were thawed and plated, usually 1 day before use
or earlier on the same day.
MATERIALS AND METHODS Primary cultures of melanoblasts and melanocytes: Primary
melanocyte cultures were made from trunk skin of neonatal
Materials: Tissue culture plastics (Nunc) and fetal calf se- a/a, m/m mice (obtained as above) and control C57BL/6J
rum (FCS) were from GIBCO Europe (Uxbridge, UK). 12-O- (a/a 1/1) mice, by procedures described previously (Svid-
tetradecanoyl phorbol acetate (TPA), cholera toxin (CT), mi- erskaya et al. 1995). In short, the skin was split with trypsin,
tomycin C, 2-mercaptoethanol (2-ME), phenylthiourea (PTU; epidermal sheets were pooled and dissociated briefly with
also known as phenylthiocarbamide) and [4-Nle,7-d-Phe]- trypsin and EDTA, and the resulting cell suspension was
a-melanocyte stimulating hormone (here abbreviated to plated on to XB2 immortal keratinocyte feeder cells. The vol-
N-MSH) were from Sigma Chemical (Poole, UK). Basic fibro- ume of culture prepared per mouse was 12 ml in six 3-cm
blast growth factor (bFGF) (bovine) was supplied by Penin- dishes for growth/differentiation experiments or 8 ml in two
sula (St. Helens, UK). All protein factor stock solutions were 5-cm dishes for immortalization. The basic growth medium
prepared in phosphate-buffered saline (PBS) with bovine se- was RPMI-1640 supplemented with penicillin, streptomycin,
rum albumin (1 mg/ml) as carrier and stored at 2708. glutamine (2 mm) and FCS (5%; 10% for cell lines once free

Figure 1.—Coat color


phenotype of misty mice. A
typical m/m mouse of the
C57BL/6J black strain is
compared to a 1/1 mouse
of the same strain. The dor-
sal view (A) shows the
slightly paler fur, normal
eyes and white feet of the
m/m mouse (right), while
ventrally (B) the typical
white belly spot is visible in
the m/m mouse (left). The
white tail-tip is also visible.
Pale fur around the nipples
(seen in 1/1 mouse, which
is female) is normal in nona-
gouti mice.
misty in Melanocytes, Platelets and Fat 383

of fibroblasts). Other supplements, where specified, were tion in a restrainer with the tip of the tail 4–5 cm below the
added to primary cultures after cell attachment or 1 day later. body. The time required for the small stream of blood to stop
Incubation was with 10% v/v CO2 at 378, which gives a suit- abruptly was taken as the bleeding time. Statistical analyses of
able pH of 7.0–7.1. bleeding times and other parameters were performed with
Serial passage of melanocytes: Cultures for the prepara- the Student’s t-test.
tion of cell lines were grown in RPMI-1640 growth medium, Platelets were counted manually (Brecher and Cronkite
with various supplements as discussed (see results). New 1950). Platelets were collected for serotonin and adenine nu-
feeder cells were added after 2–3 wk if a culture was not pas- cleotide assays as described (Swank et al. 1991). For serotonin
saged by then. Cells were subcultured by one rinse in Dul- analyses, platelets were lysed in 1 ml of distilled water and as-
becco’s PBS lacking calcium and magnesium chlorides sayed fluorometrically as described by Crosti and Lucchelli
(PBSA) and one rinse in PBSA containing 250 mg/ml trypsin (1962). Platelet adenine nucleotides were determined by a lu-
and 200 mg/ml EDTA; they were incubated until completely ciferin-luciferase system (Novak et al. 1981).
detached and resuspended in growth medium for dilution as
required. misty cell lines, once established, were grown in
RPMI growth medium with 10% FCS, 200 nM TPA and 100 RESULTS
mm 2-ME. The tyrosinase inhibitor PTU was also added at 100
mm for a few days before freezing and after thawing cell stocks General histology, deficiency of brown fat: Organs
in liquid nitrogen, and at 200 mM to the frozen stocks; this re- were compared between m/m and 1/1 C57BL/6J
duced melanin content and appeared to improve viability of
stocks. Indeed, we are now similarly using PTU for freezing of mice using sections through 1-day-old mice of both
all pigmented melanocyte lines. genotypes at similar levels. Abnormalities were found
Melan-a (a/a) and melan-c (albino, c/c) melanocytes were in only two organs. One was the skin, where the deep
grown as described (Bennett et al. 1989). Mel-18 and mel-29 portions of hair follicles were mostly well pigmented in
are melanocyte lines obtained from reversion spots on W rio/1 wild-type mice but poorly or unpigmented in neonatal
mice (De Sepulveda et al. 1995). These cells had lost the W rio
allele by somatic recombination and are thus wild-type (De misty mice, consistent with the overall m/m phenotype
Sepulveda et al. 1995). These were provided by J.-J. Panthier of diluted coat color. The other was the brown (mul-
and grown in RPMI-1640 growth medium supplemented with tilocular) fat that forms interscapular and lateral tho-
FCS (10%), 2-ME (100 mM) and TPA (200 nM). racic (axillary) aggregations or pads in mice and other
Cell proliferation and melanin assays: Triplicate cultures were rodents (Bloom and Fawcett 1986); these fat pads
plated at the specified density on 3-cm dishes for counting or
3- to 10-cm dishes for melanin assays and cultured as speci- were clearly visible in the wild-type mice (Figure 2, A
fied. Media were renewed twice weekly. Cells were harvested and C). In m/m mice they were absent, being replaced
separately from each plate by trypsinization. Triplicate cell by a thin layer of fibrous connective tissue in the inter-
counts from each suspension were made by hemocytometer. scapular location (Figure 2B) and by a compact, almost
Primary cultures for growth/differentiation experiments epithelioid layer of roundish cells in the axillary posi-
were plated in 3-cm dishes on XB2 feeder cells and were
grown in the media and for the times specified. Counts of tion (Figure 2D). It was possible that these roundish
melanocytes and melanoblasts were done on formaldehyde- cells were brown (multilocular) adipocytes lacking
fixed cells as before (Sviderskaya et al. 1995). lipid, because either immature adipocytes (Néchad
For melanin assays, at least two experiments for each cell 1986) or brown adipocytes depleted of lipid following
line were performed. Cells were plated at 1.5 3 104 cells/ml hormonal or thermal stimulation (Bloom and Faw-
and grown until they were about 75% confluent (about 7–9
days for 1/1 lines, 11–14 days for m/m lines). Melanin assays cett 1986) have a somewhat similar epithelioid ap-
were carried out as described (Friedmann et al. 1990). After pearance. No brown fat was detected in m/m mice in
removal of an aliquot for counting, the remaining cells were other expected positions either: in the perirenal area
pelleted; pellets were resuspended in 100 ml of 1 m NaOH or around the aorta and great vessels of the neck
and diluted with water (400 ml). The OD475 was measured and (Bloom and Fawcett 1986).
converted to melanin content via a standard curve using syn-
thetic melanin (Sigma). This was normalized to cell number. The outbred misty mice initially obtained seemed
To exclude photometric absorbance not due to melanin, we relatively small at birth, but after backcrossing to the in-
performed assays on melan-c albino (unpigmented) melano- bred C57BL/6J strain for several generations, the birth
cytes in the same way, and the mean absorbance per melan-c weight of m/m mice (1.44 6 0.08 g, n 5 8) was similar
cell was subtracted from the mean values for other lines. to that of 1/1 mice of this strain (1.42 6 0.02 g, n 5
Bleeding times and platelet analyses: For bleeding time and
platelet analyses, misty mice of either the C57BL/6J or the 90). Litter sizes were noted because mice in larger lit-
C57BLKS/J strain were purchased from the Jackson Labora- ters tend to be smaller. Mean sizes of the litters exam-
tory (Bar Harbor, ME) together with appropriate control ined were similar however: 2m/m, 8.38: 1/1, 6.80.
mice of the same strain, and breeding colonies of each were Body weight in older mice was not studied.
maintained at Roswell Park Cancer Institute. No significant misty melanoblasts and melanocytes in primary cul-
differences between the two misty strains were noted in any
hematological parameter. Also, no differences were noted be- ture: To study the general properties of misty melano-
tween male and female mice. Mice 6–12 wk old were used. blasts and melanocytes, primary cultures were prepared
Bleeding times were determined by tail bleeding time es- from neonatal m/m skin in “melanocyte medium” con-
sentially as described (Dejana et al. 1979; Novak et al. 1988). taining TPA and cholera toxin (CT), a cAMP agonist
A 2-mm portion of the tail was severed with a sharp razor that strongly stimulates growth and differentiation of
blade and the tail was immersed in a beaker of physiologic sa-
line at 378. Each mouse was maintained in a horizontal posi- wild-type cultures (Sviderskaya et al. 1995). In this me-
384 E. V. Sviderskaya et al.

Figure 2.—Transverse histological sections through C57BL/6J m/m and 1/1 neonatal mice at thoracic level, showing altered
brown fat and hair follicle pigmentation. In the dorsal region, the interscapular brown fat of 1/1 mice (A) is absent and replaced
by a layer of fibrous connective tissue in m/m mice (B) (straight arrows). The bases of most hair follicles were well pigmented in
1/1 neonatal skin (curved arrow, although this was difficult to show photographically), but not in m/m skin. The typical lateral
(axillary) brown fat of 1/1 mice (arrow, C) was also lost in m/m mice (D), but replaced in this region mainly by a somewhat epi-
thelioid tissue (arrow) (see text). Scale bar, 100 mM.
misty in Melanocytes, Platelets and Fat 385

of wild-type melanoblasts fell by around 90%; this was


mainly through differentiation to melanocytes, as judged
both by microscopy, and because the ratio of melano-
cytes to melanoblasts increased, for example, from
about 10:1 (14 days) to about 40:1 (21 days) with CT
(Figure 4). Conversely, the numbers of m/m melano-
cytes and melanoblasts remained steady between 14
and 21 days, either with or without CT, as did their ratio
(about 10:1). Thus, neither growth nor differentiation
of the m/m primary cultures was significantly stimulated
by CT (Figure 4).
Establishment of immortal lines from misty melano-
cytes: The deficient proliferation of misty melanocytes
caused prolonged difficulties with the establishment of
cell lines, but eventually three such lines were isolated
from misty cultures: melan-m1, melan-m2, and melan-
m3, as follows. These lines were from simultaneous but
separate primary cultures and were kept separate
throughout. All were grown with XB2 keratinocyte
feeder cells, which were replaced regularly (see mate-
rials and methods). Initially, a variety of culture sup-
plements were tested, including those used for mela-
noblasts grown without serum by Hirobe (1992).
However, under our conditions no improvement was
seen over the medium supplemented with TPA (200
nM) and CT (200 pM), which was therefore used from
3 wk onward. Poor cell growth with much cell death
continued. Moreover, microscopy of the cultures sug-
Figure 3.—Primary cultures of melanoblasts and melano-
cytes, showing atypical colony morphology in mutant cul- gested that the misty melanocytes were more darkly
tures. Wild-type (A) and m/m (B) melanocyte colonies are pigmented than normal. Because melanin intermedi-
shown by bright-field microscopy. Cultures were grown with ates can be toxic at high concentrations, the tyrosinase
TPA (200 nM) and CT (200 pM) for 3 wk before photogra- inhibitor PTU (50 mM) was added to all dishes from
phy. Wild-type cultures yield relatively large colonies of flat-
the sixth week of culture to reduce melanin synthesis.
tened melanocytes surrounded by smaller, dendritic melano-
cytes. misty melanocyte colonies are much smaller with an Growing colonies of cells were now observed. Two
abnormal morphology; flat cells are rare and many melano- months later the concentration of PTU was increased
cytes have a hyperdendritic shape. Scale bar, 150 mm. to 100 mM. After 5 mon of culture and subculture, me-
lanocytes appeared to be growing without fibroblasts,
so the medium was supplemented with 10% FCS in-
stead of 5%. Later 2-ME (100 mM) was added, and the
dium, wild-type melanoblasts proliferate well and dif- concentration of CT was reduced to 20 pM after pre-
ferentiate to give many colonies of melanocytes after liminary indications that these changes improved
3 wk (Bennett et al. 1987; Sviderskaya et al. 1995). growth. Ultimately, three independent immortal cell
The results were striking: m/m cultures differed visually lines were obtained in quantities sufficient for experi-
from control wild-type cultures more than any other mentation and freezing, although the time to this stage
mutant genotype we have studied. The m/m cultures was long in all three cases (7–9 mon). PTU and CT
contained few, small melanocytic colonies, which often were now omitted from the medium, except that PTU
consisted of only about one to four cells after 3 wk; was added for a few days before and after preparation
Figure 3 shows a larger colony. Moreover, the m/m me- of frozen stocks (a procedure we now use for all pig-
lanocytes often had a highly dendritic shape; a few mented melanocytes). The appearance of the three
such wild-type cells had this shape, but most were flat lines is shown in Figure 5.
and polygonal or bipolar (Figure 3). Cell counting con- Growth characteristics of misty melanocyte lines: Be-
firmed that wild-type cultures reproducibly produced cause melanocyte growth and responses to CT ap-
severalfold more melanocytes than m/m cultures in this peared deficient in primary misty cultures, we studied
medium with CT (Figure 4). There were also several- the growth of the three m/m lines and their responses
fold more melanoblasts in wild-type cultures when first to CT and MSH (melanocyte-stimulating hormone, an-
counted (after 14 days), either with or without CT (Fig- other cyclic AMP agonist) in comparison to three me-
ure 4). Between 14 and 21 days of culture, the number lanocyte lines wild-type at this locus (Figure 6). Growth
386 E. V. Sviderskaya et al.

Figure 4.—Markedly deficient growth and differentiation of m/m compared to wild-type (1/1) melanoblasts. Numbers of
melanocytes (j) and melanoblasts (h) are shown separately. The sum of the two values indicates total growth, while the ratio is a
measure of differentiation. Primary cultures were grown for the indicated times in growth medium (materials and methods)
with TPA (200 nM) and with or without CT (200 pM) as indicated. Data are the mean and SEM of means from three separate ex-
periments, with duplicate dishes in each experiment.

rate varied between lines of either genotype, but anin contents of three wild-type and three misty melano-
growth of all three m/m lines in standard melanocyte cyte lines and the effects of CT and MSH are shown in
medium with TPA was slower than normal; on average, Figure 7. Melanin contents varied markedly among the
wild-type lines produced fourfold more cells in 7 days. m/m lines, but in normal medium with TPA, all three
Cell yields in the presence of TPA were not significantly misty lines contained more melanin than any wild-type
increased in wild-type immortal lines by CT or MSH (a line, on average over threefold more, a highly signifi-
difference from primary cultures); in fact, greater rela- cant difference. After growth with CT, melanin content
tive proliferative responses to these agents (about 50% was significantly (twofold) higher in wild-type and 1.2-
more cells with either CT or MSH than without) were fold higher in misty lines on average (not significant);
seen in the misty lines. m/m cells cultured with CT and the latter numerical increase was due to a response by
MSH also gave higher yields than without these agents only one m/m line, melan-m3, whereas all wild-type
in the absence of TPA, possibly indicating an effect on lines responded (Figure 7). Similarly, all wild-type lines
cell survival, because cell number fell with time in con- contained more melanin after growth with MSH (1.8-
trol cultures with none of these supplements, for exam- fold on average), whereas only melan-m3 did so among
ple, from 3.00 (day 0) to 2.00 6 0.09 3 104 cells/ml in the misty lines.
melan-m1 cells grown for 7 days; parallel cultures Bleeding times and platelet analyses: A significant in-
reached 28.96 6 0.35 with TPA, 4.00 6 0.07 with CT, crease (3.3-fold) in bleeding time was noted in mice of
3.78 6 0.14 with MSH and 2.35 6 0.10 with 20 pM homozygous misty genotype (Table 1). Prolonged bleed-
bFGF (units of 104 cells/ml, mean and SEM of tripli- ing times occurred despite normal platelet counts and
cate dishes). The proliferative response to TPA at dif- normal platelet serotonin levels. Platelet ADP levels
ferent concentrations was examined in melan-m1 cells were, however, reduced to 41% of normal levels. The
and seemed normal, apart from growth rates being low small apparent decrease in platelet ATP levels was not
throughout (data not shown); the optimal concentra- significant. Resulting ATP/ADP ratios were about two-
tion was 200 nM, the same as for the wild-type line fold higher in misty platelets (Table 1). Results similar
melan-a (Bennett et al. 1987). to the reported values for 1/1 mice were obtained
Hyperpigmentation of misty melanocyte lines: The mel- when heterozygous (m/1) mice were analyzed (data
misty in Melanocytes, Platelets and Fat 387

Figure 5.—m/m and wild-type immortal melanocyte lines as seen by bright-field microscopy of unstained cells. (A) Melan-a
(1/1) mouse melanocytes for comparison. (B) Melan-m1. (C) Melan-m2. (D) Melan-m3. Cells were grown as described (materi-
als and methods), until approaching confluency. All cells are pigmented (only melanin is visible by bright-field optics); however,
the 1/1 cells are clearly palest. The three misty lines all have slightly different morphologies; some giant cells are visible in line
melan-m2 (usually a characteristic of slow-growing cells; this was indeed the slowest-growing line). Melan-m3 cells are particularly
dendritic. Scale bar, 250 mm.

not shown), indicating that the bleeding and ADP ef- growth is consistent with a developmental effect such as
fects, like the effect on coat color, are recessive. a lesion in a receptor or other signaling molecule. It
could also be consistent with a defect in an organelle
protein, as with silver, which encodes a melanosomal
DISCUSSION
enzyme (Chakraborty et al. 1996), and yet is associ-
The misty phenotype presents an unusual and in- ated with deficient growth in vitro and reduced num-
triguing pattern of biological effects. It is a spotting mu- bers of melanocytes in vivo (Spanakis et al. 1992). In
tation, which suggests an abnormality in a regulatory that case, poor cell growth was speculated to be associ-
pathway in melanocytes rather than in their function. ated with a disturbance in melanogenic pathways, possi-
Yet there is also a bleeding disorder. To date the exist- bly in superoxide metabolism (Chakraborty et al.
ence of a set of loci at which mutations affect color, 1996). A similar effect may be occurring in misty me-
bleeding time, and kidney function has been attributed lanocytes, which clearly overproduce melanin; this
to evolutionary relationships among the melanosome might well lead to accumulation and leakage from me-
(pigmentary organelle), lysosome, and platelet dense lanosomes of toxic melanin precursors such as dihy-
granule (Orlow 1995). These organelles have some droxyindole (Pawelek and Lerner 1978). This is con-
proteins in common, which are expected to include sistent with subjective impressions of improved net
the products of many of these loci. This framework growth of long-term cultures after addition of PTU to
would suggest instead that the misty locus encodes an inhibit melanogenesis.
organelle protein. Primary cultures of m/m melanoblasts and melano-
The deficient proliferation and apparently increased cytes showed no significant response to the cAMP ago-
death rate of m/m melanocytes in culture suggest that nist CT, which strongly stimulates growth and differ-
the pigmentary defects in vivo may reflect an insuffi- entiation of 1/1 murine diploid melanocytes. In
cient number of hair-follicle melanocytes. The poor general, cAMP and its agonists are well known as stimu-
388 E. V. Sviderskaya et al.

Figure 7.—Melanin contents of wild-type and misty me-


lanocyte lines. Cells were plated at 1.5 3 104 cells/ml and har-
vested at about 75% confluency, i.e., after 7 days (mel-18 and
-29), 9 days (melan-a), 11 days (melan-m1 and melan-m3)
and 14 days (melan-m2) in culture. Cells were grown in RPMI
growth medium (materials and methods), with TPA (200
nM) and either with or without CT (200 pM) or MSH (1 nM).
Shown are means and SEM of six cultures from each cell line
(triplicate dishes from each of two experiments). To exclude
photometric absorbance not due to melanin, we subtracted
the mean absorbance per melan-c (albino) melanocyte from
the values shown here (materials and methods). There was
variability among individual lines, but the difference in mean
melanin content between misty and wild-type lines was signifi-
cant by Student’s t-test under all conditions: TPA only (P ,
Figure 6.—Proliferation of wild-type and misty melanocyte 0.01), TPA with CT (P , 0.02) and TPA with MSH (P , 0.05).
lines. Cells were plated at 1.5 3 104 cells/ml and grown in The increase in overall mean melanin content with CT or
RPMI growth medium with supplements as shown: TPA (200 with MSH was highly significant for wild-type lines (P , 0.01)
nM), CT (200 pM) or MSH (1 nM). Cells were harvested and but not significant for misty lines.
counted at about 75% confluency (at times varying between
cell lines). To facilitate comparison, doubling times were ob-
tained from cell counts for each line on the assumption of ex-
ponential growth (adequately correct under these condi- PKA substrate such as a transcription factor. Another
tions) and were used to calculate the expected cell number class of explanation is suggested by the observations on
after 7 days (shown). Wild-type lines melan-a, mel-18, and
mel-25 were compared to the three misty lines. Data are the
platelets (see below). Whatever the mechanism, the ob-
means and SEM of means from six separate experiments per served partial restoration of responsiveness to CT in im-
genotype (two for each cell line, with triplicate dishes in each mortal m/m melanocytes, especially noticeable in line
experiment). Differences in cell proliferation between misty melan-m3, could result from a reversion, compensating
and wild-type lines were significant by Student’s t-test under
all three conditions (P , 0.05).
TABLE 1

lators of both melanogenesis and dendrite formation Hematological parameters of normal (1/1)
and misty (m/m) mice
in pigment cells, and are believed to act through pro-
tein kinase A (PKA), at both the post-transcriptional Parameter Normal (1/1) misty (m/m)
(e.g., Wong and Pawelek 1975; Preston et al. 1987)
and transcriptional levels (e.g., Bertolotto et al. Bleeding time (min) 4.0 6 0.63 (11) 13.2 6 0.83 (16)*
Platelets/ml (109) 0.62 6 0.04 (6) 0.55 6 0.03 (6)
1996). This finding raises the specific possibility of an
Serotonin
abnormality somewhere in the cAMP signaling path- (mg/109 platelets) 1.57 6 0.17 (8) 1.35 6 0.13 (8)
way. The overproduction of melanin and dendrites by ATP (mmol/1011
m/m cells suggests overactivity of some component, platelets) 4.66 6 0.27 (11) 3.67 6 0.34 (11)
leading to maximal or near-maximal activity of the ADP (mmol/1011
pathway and hence lack of stimulation by CT. This platelets) 3.22 6 0.24 (11) 1.32 6 0.20 (11)*
could happen if misty encoded a subunit of adenylate ATP/ADP 1.44 2.78
cyclase (such as an inhibitory a subunit of the GI, GQ or Data are the mean and SEM of values from the number of
GZ families; Wilkie et al. 1993), a subunit of PKA, or a mice in parentheses. *P # 0.001.
misty in Melanocytes, Platelets and Fat 389

mutation or change in expression of the misty gene platelet storage pool deficiency patients having mark-
product or an interacting one. edly lowered ADP accompanied by mild reductions in
It is possible that overproduction of cAMP in mul- serotonin. Additional studies will be required to deter-
tilocular adipocytes (brown fat cells) could lead to the mine whether the lowered platelet ADP in misty plate-
delipidation or atrophy of this tissue, because cAMP lets is strictly in the dense granule storage ADP pool, as
appears to be a signal for lipolysis in this tissue, and would be expected for a platelet storage pool defi-
atrophy can follow prolonged lipolysis (for review, see ciency syndrome, or whether the mutant platelet meta-
Nicholls and Locke 1984). Interestingly, it was re- bolic pool is also affected.
cently reported in an abstract that older m/m mice of An interesting related issue is whether ADP levels
the C57BL/6 strain also have reduced amounts of are lowered in melanocytes and fat as they are in plate-
white fat and (from the second week) reduced body lets. If ADP is depressed and if cAMP production is like-
weight (Truett et al. 1996). We did not detect this be- wise modulated by ADP in these cells as it is in platelets,
cause we examined only neonatal mice of this strain. It a reasonable explanation for the overproduction of
seems plausible that the misty mutation may affect both melanin and dendrites in melanocytes and for the de-
brown and white fat by similar mechanisms. lipidation or atrophy of mutant brown and white fat is
The observation of reduced levels of platelet ADP available. These possibilities are under investigation.
provides a likely explanation for the prolonged bleed-
ing in misty mice. It is well known that ADP is an impor- We are indebted to Jean-Jacques Panthier (Ecole Nationale
Vétérinaire d’Alfort) for melanocyte lines mel-18 and mel-29, to
tant physiological agonist that stimulates platelet aggre- Simon Hill and Madonna Reddington for skilled technical assis-
gation (Holmsen 1989). ADP released from the tance, and to George Mewis for expert preparation of histological
platelet dense granule pool during aggregation is in sections. This work was supported by Wellcome Trust grants 036603/
turn a potent platelet agonist that interacts with an Z/92/Z and 046038/Z/95/Z, by grant HL-31698 from the National
ADP receptor on adjacent platelets to stimulate them Heart, Lung and Blood Institute and by Roswell Park Cancer Insti-
tute core grant P30 CA 16056.
further and ultimately to form a platelet plug that ar-
rests bleeding. Our data rule out thrombocytopenia
and platelet serotonin deficiency, which cause inher- LITERATURE CITED
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