Download Brief Communications - Peromyscus Genetic Stock Center

Brief Communications
S. K. Teed, J. P. Crossland, and W.
D. Dawson
where a small breeding stock was maintained until 1981, when the stock was expanded for further study.
trephination needle. Dummy pellets were
implanted into control animals. The area
of the implant was then shaved. Hair regrowth was apparent about 3 days later.
Materials and Methods
Ashy deer mice were outcrossed to a wildtype stock of P. maniculatus bairdii, BW, to
improve fertility, and the mutant was reAshy deer mice (Peromyscus maniculatus) covered in the F2. Genetic segregation was
were first discovered about 1960 in a wild
analyzed by chi square in these crosses
population from Oregon. Although indistinand subsequent backcrosses. The husguishable from the wild type at weaning, ashy
bandry procedures used were similar to
deer mice become progressively grayer with
those for laboratory mice. Animals were
subsequent molts. The trait is inherited as an
reared in a controlled environment at 22autosomal recessive and the symbol ahy is
25°C on a uniform 16:8 light: dark cycle.
assigned for the locus. The trait is distinctly
Animals were examined at 2- to 7-day
manifest by 6 months of age, at which time
intervals from birth until 6 months of age
homozygotes have white hairs on the muzzle
to ascertain details of phenotypic develand at the base of the tail. The amount of
opment. Significant progressive changes
white gradually increases with age, but dein development of the trait were recorded
velopment varies greatly among animals.
at the time of first detection. A photoSome become virtually all white by 18 months. graphic record was also made of selected
Implants of melanocyte-stimulating hormone
animals.
induced production of pigment in depigThe coat was examined by naked eye to
mented portions of the coat, indicating that
observe gross pigmentation patterns. Hairs
viable melanocytes were present. The ashy
from the head, mid-back and rump of mudeer mouse model may be useful for further
tant and control deer mice were examined
study of melanocyte function.
under a dissection microscope. Selected
mid-dorsal hairs were suspended in glycThe ashy mutant deer mouse was first isoerol and permount, mounted on slides and
lated about 1960 by Ralph R. Huestis (perexamined with a compound light microsonal communication, 1964) from a wild
scope. The three categories of hairs chopopulation of Peromyscus maniculatus rub- sen were pigmented hairs from wild-type
idus inhabiting the sand dunes near Alsea animals, pigmented hairs from ashy deer
Bay, Oregon. Huestis noted that these anmice, and unpigmented hairs from ashy
imals became progressively grayer with
deer mice. Representative hairs were phoeach subsequent molt. His initial analysis tographed.
of breeding data indicated that the trait,
Melanocyte-stimulating hormone (MSH)
originally called "dunes ashy," is inher- implants were made according to the
ited as a recessive. However, the ashy mu- method of Geshwind (1972). One-mg peltation was never formally reported in the
lets of beeswax and sesame oil impreggenetic literature. Six pairs of ashy deer
nated with 0.05 mg of a-melanocyte-stimmice from the Huestis stocks were sent to ulating hormone were subcutaneously
the Peromyscus Genetic Stock Center at
implanted into the unpigmented forethe University of South Carolina in 1965,
heads of mature ashy deer mice using a
Description of Trait
Ashy Peromyscus appear normal as juveniles. By 2 months of age, some animals
destined to become ashy develop paler
than normal ankles above the white foot
typical of this genus (Figure 1). Another
harbinger of the ashy trait in many cases
is a nose blot, formed by depigmentation
of the skin surrounding the distal end of
the rostrum that leaves a pigmented spot
at the tip. Progression of the trait (Figure
2B-G) follows a typical pattern: between
80 and 120 days, the first appearance of
gray hairs on the rostrum occurs, and gray
begins to extend upward on the limbs (Figure 2B), but some animals still are indistinguishable. By 120 to 180 days, more than
90% of homozygotes show some graying
on the muzzle and base of the tail (Figure
2C). At 6 to 9 months, virtually all animals
exhibit some manifestation of the trait. Gray
on the rostrum and rump becomes more
extensive and prominent (Figure 2D). The
area of graying tends to extend backward
from the face, forward from the rump, and
upward on the legs with each subsequent
molt; the interscapular region of the back
is thefinalarea to lighten. By 9 to 12 months
(Figure 2E) most homozygotes show a distinctly "ashy" aspect, hence the locus designation. After 1 year, depigmentation continues slowly. The face and rump are
mostly white, but the dorsum is partially
pigmented. Many ashy animals do not progress beyond this stage despite increasing
age, but some continue to lose pigment
(Figure 2F) until the animal is virtually all
white with a scattering of pigmented hairs
on the back. The eyes remain fully pigmented in the stage called "black-eyed
309
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Coat Color Genetics of
Peromyscus. I. Ashiness, an
Age-Dependent Coat Color
Mutation in the Deer Mouse
706
Figure 1. Early evidence of the ashy phenotype: (A) Ankle band (arrow) seen in wild type. (B) Absence of
band and graying of ankle in ashy type. Both animals are about 60 days old.
Table 1. Inheritance of ashy mutant deer mice
Analysis"
Phenotypes
Mating types
Female
Male
Number
of
matings
Wild-type
Ashy
Female
Male
Female
Male
Segregation
Sex
Interaction
Total
Ratio x2
Ratio X2
Ratio> X2
Ratio
X2
+/ahy x +/ahy
2
Observed
Expected
8
11.25
15
11.25
2
3.75
5
3.75
3:1
0.044*
1:1
3.333*
1:1
0.093'
3:3:1:1
3.473'
+/ahy x ahy/ahy
1
Observed
Expected
1
1.75
2
1.75
1
1.75
3
1.75
1:1
0.143"
1:1
1.286"
1:1
0.143*
1:1:1:1
1.572'
5
Observed
Expected
18
13.25
13
13.25
14
13.25
8
13.25
1:1
1.528'
1:1
2.283*
1:1
0.019*
1:1:1:1
3.830'
+ / + x ahy/ahy
3
Observed
Expected
32
31
30
31
—
ahy/ahy x ahy/ahy
5
Observed
Expected
0
0
ahy/ahy x +/ahy
1"
0
• All nonsignificant at P < .05.
*df = 1.
'df = 3.
* One animal had not expressed the ashy trait by 8 months of age.
3 1 0 The Journal of Heredity 1990:81(4)
0
0
0
0
1:0
-
1:1
0.065*
—
—
—
32
33.5
35
33.5
—
—
1:1
0.134'
0:1
—
—
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whites" (Figure 2G). Skin pigmentation in
the ears, tail, and scrotum also remains.
Animals remain vigorous, healthy, and fertile, and hearing and eye pigmentation apparently are unaffected. After 2 years, decline occurs at the normal rate, with no
evidence of premature aging other than in
the coat. Some ashy animals survive more
than 4 years. Nonashy wild-type deer mice
(Figure 2A) live 4 or more years without
conspicuous coat graying.
Individual pigmented mid-dorsal hairs
from ashy animals exhibited obvious microscopic differences from wild-type hairs
(Figure 3, A and B). These ashy hairs had
anomalously shaped pigment granules,
conspicuously thinner cuticles, and irregular spacing of granules. White hairs from
ashy deer mice were completely devoid of
pigment granules (Figure 2C).
Two ashy animals with MSH implants in
their unpigmented foreheads produced
pigmented hairs over the area of the implant (Figure 4), whereas another with a
dummy implant continued to produce unpigmented hair, suggesting that the hormone stimulated pigment production by
melanocytes in the follicles. Examined microscopically, pigmented hairs above
treated implants displayed absence of
agouti bands and phaeomelanin. MSHstimulated hairs also showed more irregular pigment granules than shown in wildtype pigmented hairs. The two wild-type
control animals with sham implants continued to produce normally pigmented
hairs.
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Lite*
Figure 2. (A) Wild-type adult. (B-F) Typical development of the ashy phenotype. (G and H) Extreme depigmentation observed in some animals.
Inheritance
Ashiness is inherited as an age-dependent, variably expressed, autosomal recessive trait (Table 1). Since young ashy
deer mice are virtually indistinguishable
from the wild type, the phenotype can not
be scored reliably in some animals until
6-8 months of age. The degree of expression and time of first appearance of gray-
ing varies considerably among animals and
sibships, suggesting that physiological
factors or modifier genes may play a role
in expression. The distribution of pigment
loss on the body is symmetrical. One pre-
Brief Communications 3 1 1
The genetic locus is designated ahy. Genetic linkage and interactions for ashiness
have not been explored.
Discussion
Figure 3. Mid-dorsal hairs photographed under 40 x magnification. (A) Wild type: fully pigmented, normal
hairs. (B) Ashy: totally depigmented hairs. (C) Ashy: irregularly pigmented hairs.
sumptive homozygous recessive animal
never developed the ashy phenotype, although it was retained longer than 8
months, suggesting that the gene is not
100% penetrant. A revertant also could account for this exception.
Ashy heterozygotes are indistinguishable from wild-type animals at maturity,
with the possible exception that some may
exhibit a trace of the nose blot effect noted
above. The gene is not sex-linked, because
ashy females mated to wild-type males
produced both ashy and wild-type male
and female progeny. There was no evidence of sex influence on the phenotype
or time of expression of the trait.
Figure 4. Repigmentation of coat above location of MSH implant (arrow).
3 1 2 The Journal of Heredity 1990:81(4)
In the house mouse (A/us musculus domesticus), the autosomal dominant Ga gene
results in graying with age (Silvers 1979).
In this case, the mice begin producing unpigmented hair by 10 months; the earliest
onset is 6 months. The gray first appears
on the ventrum, then on the rump and
face, with the mid-dorsum being the last
area affected. Individual hairs are either
fully pigmented or totally devoid of pigment. The milk-transmitted murine leukemia virus has been implicated in graying
with age in house mouse (Morse et al.
1985). Ashiness in Peromyscus is phenotypically similar to Ga, but differs in several respects. Animals that show graying
associated with age (Ga) exhibit bilateral
asymmetry, higher mortality than nongray
animals (with increased incidence of
splenomegaly and neoplasms), frequently
misshapen vibrissae, and a dominant mode
of inheritance. None of these effects is typical of ashy Peromyscus. Nevertheless, the
possibility that ashiness is the product of
a gene-virus interaction or is the consequence of the insertion of a transposable
element cannot be excluded. Cross-fostering of caesarian-derived offspring may
provide evidence for or against lactational
transmission. Ashiness is not homologous
with ashen (as/i) in the mouse, which is
dilute-like and not age-dependent (Lane
and Womack 1979).
A trait called "grizzled," described in P.
maniculatus by Sumner (1928), resulted in
the "replacement of colored hairs by white
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B
The loss of hair pigment with increasing
age occurs in many mammals, including
humans. The rate at which depigmentation occurs varies among individuals and
is generally assumed to result from progressive necrosis of melanocytes. Polygenic factors may play a role in normal
graying of humans and other species
(Keogh 1965). However, early and pronounced graying may result from the action of single genes, as in the case of dominant gray in horses. Horses with the gray
(G) gene have pigmented coats as colts,
but lose color with subsequent molts,
eventually appearing white. Homozygotes
begin graying earlier and to a greater extent than do heterozygotes. Both homozygotes and heterozygotes retain skin pigmentation where the hair was originally
pigmented.
From the Department of Biological Sciences, University of South Carolina. A portion of this study is based
on an honors thesis presented to South Carolina College by S.K.T. The animals used in this project were
housed in the Peromyscus Genetic Stock Center, which
is supported in part by National Science Foundation
Grant BSR-8419860. The authors gratefully acknowledge the contribution of G. Whitley, who, as an undergraduate student, nurtured the ashy mutant through
a critical period and prevented its extinction. They
also thank A. Lawson and C. Joyner for providing excellent animal care, and F. Bolander and L. Kwarsick
for assistance with the MSH implants. Photography is
by C. Cook. Address reprint requests to Dr. Dawson,
Department of Biological Sciences, University of South
Carolina, Columbia, SC 29208.
References
Bronson FH, 1969. Melanocyte-stimulating activity following adrenalectomy in deermice. Proc Soc Exp Biol
Med 130:527-529.
Geshwind 1,1972. The effect of melanocyte stimulating
hormone on coat color in the mouse. Rec Prog Hormone Res 28:91-130.
Keogh EV, 1965. Rate of greying of human hair. Nature
499:877-878.
Lane PW and Womack JE, 1979. Ashen, a new color
mutation on chromosome 9 of the mouse. J Hered 70:
133-135.
Morse HC, Yetter RA, Stimpfling JH, Pitts OM, Fredrickson TN, and Hartley JW, 1985. Greying with age in
otophora as the fusion partners. There were
several reasons for choosing these species.
Silvers WK, 1979. The coat colors of mice. New York:
First, the technique for protoplast culture
Springer-Verlag.
of these two species has been established,
Sumner FB, 1928. Observations on the inheritance of
and it appears that the same method is
a multifactor color variation in white-footed mice
appropriate for both species (C.-C. Chen,
{Peromyscus). Am Nat 62:193-206.
unpublished results). Second, they can be
crossed sexually and the hybrids grow
well,2 and so it can be anticipated that their
genomes are compatible in somatic hybrids. Third, the interphase nuclei of these
Somatic Hybridization
two species differ in regard to the amount
of heterochromatin,8 and this difference
between Nicotiana sylvestris
can be used as a basis to determine the
and N. otophora without the
nuclear condition of protoplasts after the
Application of Selection
induction of fusion. Fourth, the karyotypes
of these two species are markedly differF.-M. Lee and C.-C. Chen
ent,2 and so it is possible to determine the
Mesophyll protoplasts of Nicotiana sylvestris genome constitution of plants obtained
and N. otophora were mixed at equal density, from somatic hybridization.
mice: relation to expression of murine leukemia viruses. Cell 41:439-448.
treated with polyethylene glycol (PEG), and
then cultured in medium with no selection
pressure against parental cells. Cytological
examination of protoplasts after PEG treatment revealed 6.5% homokaryocytes and
4.5% heterokaryocytes. Despite the low frequency of heteroplasmic fusion, about 33%
of calli differentiated into hybrid plants that
contained the genomes of both parents. The
ploidy levels of the somatic hybrids varied,
and the frequencies of plants with SSOO,
SSSSOO, SSOOOO, and SSSSOOOO genome constitutions were 72.1%, 8.8%, 7.4%,
and 11.8%, respectively. Aneuploid numbers
and chromosome structural changes were
detected in somatic hybrids. The successful
recovery of somatic hybrids without deliberate selection is attributed to the differential
responses of parental protoplasts to PEG
treatment and culture and to genetic complementation in the hybrids.
Somatic hybridization can overcome barriers to sexual crosses and therefore is a
powerful tool in fundamental research and
plant breeding. Because the formation of
heterokaryocytes is generally low after the
induction of protoplast fusion, selection of
hybrid cells becomes an essential procedure in somatic hybridization. Consequently, various methods of selection have
been developed.3
There have been reports that somatic
hybrids can be obtained without the application of selection.3 The calli of some
interspecific hybrids are often more vigorous and grow faster than does the callus
of either parent."-' 4 However, this may not
be the only explanation for all the cases
reported.
We investigated this problem, using protoplasts of Nicotiana sylvestris and N.
We report the nuclear condition of protoplasts after treatment of the protoplast
mixture of these two species with polyethylene glycol (PEG) and the genome constitution of plants recovered in the absence of artificial selection. We discuss the
reason for our successful recovery of somatic hybrids. To our knowledge, somatic
hybridization between these two Nicotiana
species has not been reported.
Materials and Methods
Plant Material
Seeds of Nicotiana sylvestris Spegazzini &
Comes (2n = 2x = 24) and N. otophora
Grisebach (2n = 2x = 24) were supplied
by Dr. V. A. Sisson of the Tobacco Research Laboratory, USDA, Oxford, North
Carolina. They were sterilized in 1% sodium hypochlorite for 20 min, washed
thoroughly in sterile distilled water, and
germinated on an agar-solidified medium
containing Murashige and Skoog salts9 and
3% sucrose. We maintained and propagated the plants in vitro as described by Negrutiu and Mousseau.10
Protoplast Isolation, Fusion, and
Culture
We used the upper two or three fully expanded leaves of plants cultured in vitro
as the source of protoplasts. Protoplasts
of the two species were isolated separately, according to the method of Huang and
Chen.4 The protoplasts were washed once
with W5 solution7 and resuspended in this
solution at a density of 2 x 10s/ml.
For protoplast fusion, we followed the
PEG method of Kao et al.5 with some modifications. Protoplast suspensions of the
two species were mixed at equal volumes.
Brief Communications 3 1 3
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hairs in certain parts of the pelage." The
mode of inheritance was reported as a
"complex dominant," but a consistent pattern of segregation was elusive. The grizzled variant no longer exists in laboratory
deer mouse stocks. Ashy and grizzled mice
phenotypically were very similar, according to Huestis (personal communication),
who observed both traits. The possibility
exists that the ashy and grizzled traits are
manifestations of the same mutation, which
may occur as a polymorphism in Pacific
coastal deer mouse populations.
A number of other known variants produce gray or white patches or bands in
older Peromyscus. The genetic basis for
these, if any, is unreported, and no systematic study of them has been conducted,
although visually these traits are much less
symmetrical in their expression than is
ashiness.
Deer mice with adrenal tumors have elevated MSH, which produces hyperpigmented melanistic animals (Bronson
1969). The coat pigmentation of laboratory
house mice also can be manipulated with
MSH (Geshwind 1972). The implantation
of MSH-laden pellets in the study reported
here produced pigmented hairs in the depigmented areas of ashy deer mice, indicating that functional melanocytes were
present in the follicles. Therefore if premature melanocytic necrosis is the basis
of the ashy condition, apparently it is incomplete.