Download Mesenchymal Dysplasia: A Recessive Mutation

Mesenchymal Dysplasia: A Recessive
Mutation on Chromosome 13 of the Mouse
H. O. Sweet, R. T. Bronson, L. R. Donahue, and M. T. Davisson
Mesenchymal dysplasia (mes) is a new autosomal recessive mouse mutation that
alters normal growth of mesenchyme-derlved tissues and provides a new mouse
model for studying connective tissue development and defects. Mutants are characterized by preaxial polydactyly of all four feet, a shortened face, wide set eyes,
domed head, and a shortened kinky tail. Multiple skeletal defects are seen in alizarin-stained specimens. Hlstologlcally, areas of mineralization are found In tendons. Mutants also have increased musculature In the shoulders and hips and
decreased peritoneal fat. Salivary glands, testes, and kidneys are smaller than in
llttermates. Mesenchymal dysplasia has been mapped to mouse chromosome (Chr)
13. These mapping crosses also confirmed that the Purklnje cell degeneration (pcd)
mutation is on Chr 13.
From The Jackson Laboratory, 600 Main St., Bar Harbor, Maine 04609-1500 (Sweet, Bronson, Donahue, and
Davisson) and Tufts University, School of Veterinary
Medicine, Boston, Massachusetts (Bronson). We dedicate this paper to Dr. Margaret C. Green. This work
was supported by grants BIR 89-15728 from the National Science Foundation, P40 RR01183 and Cancer
Core Center grant CA34196 from the National Institutes
of Health, and a gift from the Eleanor Naylor Dana
Charitable Trust. The antlserom (UB2-495) to IGF-1 was
a gift from the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and
Kidney Diseases, the National Institute of Child Health
and Human Development, and the U.S. Department of
Agriculture. We thank Belinda Harris, Ann Hlgglns, and
Prlscllla Jewett for hlstologlcal preparations, W. a
Beamer lor helpful discussions, Unda Nelesld for
manuscript preparation, and Drs. Margaret C. Green
and Kevin Flurkey for critical review of the manuscript.
Address reprint requests to H. 0. Sweet at the address
above.
Journal of Heredity 1996^7^7-95; 0022-1503/96/J5.00
Inherited malformation syndromes in mice
provide valuable model systems for understanding gene defects creating similar
human conditions (Winter 1988). With the
rapidly expanding mouse genetic map, the
structural genes for many mutations causing such syndromes are now being identified. Thus, each new mouse mutation
that causes congenital malformations provides the potential of identifying the gene
affected in comparable human conditions.
In addition, mouse mutations that cause
developmental abnormalities provide model
systems to study basic mammalian development. We describe here a new recessive
mutation in the mouse that affects many
mesenchyme-derived tissues. The mesenchymal dysplasia(mes) mutation causes
multiple skeletal anomalies, including preaxial polydactyly of all four feet, a shortened
and wider than normal face with wide set
eyes, a domed head, a broad thoracic region, and a kinky tail. The most striking features of the mutant are excess skin and increased musculature in shoulder and hip
regions. Failure of the testes to descend results in sterility in virtually all males. Mesenchymal dysplasia maps to mouse chromosome (Chr) 13 near four genes that involve growth regulation.
Materials and Methods
Mice
All mice were reared and all genetic breeding studies were carried out in the Mouse
Mutant Resource at The Jackson Laboratory (Davisson 1990). Mice were maintained in a modified barrier mouseroom.
The room with a filtered air supply is
maintained at 68°F, 40-50% humidity. Mice
are caged in polycarbonate cages (51 in2)
on sterilized white pine shavings. All boxes are covered with a flat filter. Water supplied to the animals is both acidified and
chlorinated with a pH of about 2.5, residual chlorine content of 12 to 18 ppm. The
water treatment program suppresses the
growth and spread of Pseudomonas sp.
The strain is maintained on the 96W diet
formulation manufactured by Emory Morse
Company, Guilford, Connecticut (protein
22.48%, fat 7.13%, fiber 2.58%, ash 4.57%, Ca
0.22%, P 0.67%). Descriptions of the mutant
genes that were used in tests for allelism
may be found in Green (1989) or in MGD
(1994). Genetic crosses are described in detail in Results.
Phenotypic Studies
Eleven pairs (four female pairs, seven
male pairs) of mes/mes and littermate controls (+/?) were weighed at weekly intervals (to the nearest 0.1 g) for a 4 week
period. The sample included three different groups of mice starting at 3, 5, and 11
weeks of age, respectively. Seven of these
pairs (three female, four male) and two
known heterozygotes (+lmes) were euthanized by CO2 and necropsied. Their organs were weighed to the nearest 0.1 mg,
tissues saved for histology, skins mea-
87
sured, and carcasses saved for skeletal
preparations. Skins were measured by laying them flat on paper towels and measuring length and width in millimeters using
a standard metric ruler. Alizarin-stained
whole skeleton preparations were made
using the method described by Green
(1952).
Histology
Tissues for most histological examinations
were fixed in formal-acid-alcohol or
Bouln's solution. Bones were demineralized for several weeks in Bouin's solution.
Representative paraffin sections of all tissues from six adult mes/mes mice were
stained with hematoxylin and eosin
(H&E). Multiple sections of brain from one
mutant adult were stained with H&E; replicate brain sections were stained with luxol fast blue and cresylecht violet. For histological examination of testicular tissue,
testes were fixed for 1 h in 2% glutaraldehyde and 1% paraformaldehyde in 0.1 M
sodium cacodylate buffer, embedded in
JB4 resin, sectioned at 1 mm, and stained
with H&E for general examination and
with periodic acid-Schiff (PAS) for analysis
of acrosome development. Histological
sections were examined from testes of two
1 month old and two 2 month old pairs of
mes/mes mutants and littermate +/? controls.
Body Composition Methodology
The amounts of water, fat, protein, and
mineral that compose the body mass were
measured using a biochemical protocol
previously described (Donahue and Beamer 1993). Briefly, four mutant and three
normal +/? mice 8 weeks of age were fasted overnight and blood samples were collected by retro-orbital bleeding. Mice were
euthanized by exposure to CO2 and then
shaved of all hair. Gastrointestinal tracts
were emptied of food contents, carcasses
weighed, tail lengths measured, and mice
were stored frozen at -4°C until analyzed.
Deep frozen (liquid Nj) carcasses were
pulverized, lyophilized, and dry weights
obtained. Body water data were derived
from the differences between fresh carcass and lyophilized weights. Entire lyophilized samples were homogenized to uniform consistency and aliquots repeatedly
extracted with chloroform:methanol (3:2,
v/v) to remove lipid; the defatted pellet
was weighed. The difference between lyophilized mass and defatted pellet was the
amount of fat. The defatted pellet was
ashed overnight at 600°C and the ash
weighed to determine the amount of min-
8 8 The Journal oi Herecfity 1996:87(2)
eral. The difference between the defatted
pellet and ash weight yielded the amount
of protein. These data were corrected for
aliquot size to obtain the final absolute
amounts of fat, protein, and mineral present in each carcass. The data are expressed as a percent of total carcass
weight and were analyzed without arcsine
transformation because the range of percentages within a given data set was less
than 40 (Little and Hills 1975).
first analyzed for main effects using ANOVA. If significant main effects were identified, individual means were compared by
Fisher's least significant difference (LSD)
test or by Student's t test. Differences were
considered significant at p < .05. Recombination estimates were calculated using a
computer program for intercross data
(Green 1985).
Hormone Measurements
Serum insulinlike growth factor I (IGF-1)
levels in seven mes/mes and six +/? littermates (genders combined) were analyzed
by radioimmunoassay (R1A). The R1A is
composed of (1) antibody (UB3-189) produced by Underwood and Van Wyk and
obtained from the National Hormone and
Pituitary Hormone Program; (2) recombinant human IGF-I (rhIGF-I) from Bachem
(Torrance, California); and (3) 12S1-IGF-I
from Amersham (Arlington Heights, Illinois). Serum samples were prepared for
R1A of IGF-I by extraction with acid-ethanol
followed by neutralization (Donahue and
Beamer 1993).
Serum IGF binding proteins (IGFBPs)
were measured by Western Ligand Blotting as described previously (Donahue et
al. 1990). Briefly, sodium dodecyl sulfatepolyacrylamide gel electrophoresis was
performed according to Laemmli (1970)
on serum proteins separated under nonreducing conditions in 12.5% acrylamide
gels at constant current (36 mA) for 6 h.
The separated proteins were transferred
to nitrocellulose using the method of Towbin et al. (1979) and then washed in 3%
NP40, blocked with 1% BSA in TBS, and
rewashed in 0.1% Tween. Electroblotted
proteins were incubated for 24 h at 4°C
with 200,000 cpm 12SI-IGF-i and then exposed to Kodak XOMAT AR film for 4-6
days at -70°C. Radiolabeled IGFBPs on
the autoradiograms were confirmed by
l4
C-labeled molecular weight standards
(Sigma, St. Louis, Missouri) and then
graphed with a CliniScan 2 Scanning Densitometer (Helena Laboratories, Texas).
To uniformly quantify the IGFBPs, paper
densitometric peaks were cut out and
weighed. The amount of each IGFBP present in sera of mes/mes and +/? mice was
expressed in mass units for statistical
evaluation of differences between genotypes.
Origin and Description
Mesenchymal dysplasia arose in and was
originally maintained on the inbred CBA/J
background (Sweet and Bronson 1988).
Reproductive performance of the CBA/J
strain (Les 1991) made it difficult to continue inbreeding or to maintain this mutation on the CBA/J background. Therefore, the original heterozygous +/mes
male parent was outcrossed to a B6C3Fea/a (C57BL/6JLe x C3HeB/FeJLe-a/a) hybrid female. Having this mutation on the
B6C3Fe-a/a hybrid background has several advantages: it permits use of the technique of ovarian transplantation for ease
of maintenance; litter size is increased, reducing the risk of losing the mutation, and
the homozygotes produced are hardier
and live a normal life span. The mutation
is maintained in the Mouse Mutant Resource by repeated backcrossing to the
B6C3Fe-a/a hybrid background. Mice from
both backgrounds were used to characterize the phenotypic effects of the mutation;
they did not differ significantly.
Statistical Analysis
Descriptive statistics were computed for
all data by genotype and sex. Data were
Results
The first mutants submitted to the Deviant Search Program were recognized at
weaning age by preaxial polydactyly of all
four feet, dome-shaped head, white belly
spot, and kinky tail. The parents of this
litter, a sibling pair of CBA/J, produced a
total of 13 progeny in three litters, of
which four abnormal and seven normal
offspring were classified. The growth of
the affected animals was retarded compared to normal siblings and most died
before 40 days of age. On both the original
background and the present B6C3Fe stock,
the mes/mes phenotype is characterized
at birth by preaxial polydactyly of all four
feet, a shortened face, wide set eyes,
domed head, and kinky tail. The overall
body configuration of the mutant between
birth and weaning is shorter than that of
the normal sibling. The thoracic region is
broader than normal. Despite the shorter
body, internal viscera are accommodated
within the body cavities without causing a
bulging of the sides such as is seen in homozygous brachymorphic (bm/bm) mice.
Figure 1. Mutant mes/mes (right) compared to +/? littermate (left). Note folds of skin on the face and the
apparent small size of mutant eyes because of the surrounding excess skin. The mutants shown are on the original
CBA/J genetic background.
A few severely affected homozygotes display kyphosls in the shoulder region. No
scoliosis Is observed. The skull may have
either a small frontal dent or slight bulge.
Eyes are wide set and flattened rather
than bulging. The entire head appears
foreshortened and wider (see Figure 1).
There are no externally visible mandibular
defects, cleft lip, or cleft palate. Rear legs
are adducted to the sides at right angles
to the spinal column. Tails are thickened,
shortened, and kinked. Mutants may have
a white belly spot. By 5 to 6 weeks of age,
the affected animal appears larger than its
normal littermates. Folds of skin obscure
the eyes and cover the joints of the legs.
Overall, the skeletal frame of the mutant
appears unable to accommodate the ex-
Figure 2. Alizarin-stained skeletons of thorax of mes/mes (right) and +/? (left) llttermates. Ventral view showing
abnormal sternum. Note that although the mutant thorax seems smaller it Is wider In proportion to length than
the normal.
Figure 3. Alizarin-stained skeletons of left hind foot
of mes/mes (right) and +/? (left) littermates Illustrating
extra preaxlal toe (left arrow) and extra bone spur
(right area) abnormalities of the mes/mes mouse.
cessive amount of skin. Although this results in the appearance of disproportionately shortened long bones in the extremities, alizarin preparations show normal
long bone proportions. Homozygous mes/
mes adults have visibly thickened, tough
foot pads. Most homozygotes fail to reproduce (see Reproduction section). The
mes/mes homozygote is behaviorally different from its normal littermates in that
it is extremely docile.
Morphology
Measurements of the alizarin-stained skeletons showed no differences in length or
width of long bones between mes/mes and
+/? controls. Caliper measurements of
scapula, humerus, ulna, tibia, fibula, and
skull length and width showed no significant differences between mutant and control mice (data not given). No overt hydrocephalus was seen in any of the mutants
examined histologically, despite the slight
doming of the skull visible externally. Although the width between the eye sockets
in mes/mes adult mice was consistently
greater than in controls, no extra interfrontal bone was found. The mean interocular width in eight mes/mes mutants was
1.00 cm while that of seven +/? adults was
0.65 cm. The most obvious skeletal abnormalities were seen in the thorax (Figure 2)
and legs (Figure 3). All affected mes/mes
mutants exhibited fusions of sternebrae
and an abnormally shaped manubrium,
Sweet et al • Mesenchymal Dysptasia 89
about 6 weeks of age. After 6 weeks of age
the mutants weighed more than their normal littermates. Within individual litters
the differences were not always significant
(data not shown).
Figure 4. Cross sections of sacral spine and epaxial musculature from (a) a wild-type mouse and (b) a mes/mes
mouse (both 5 months old), photographed at the same magnification. Note the dramatic hyperplasla of muscle
fibers In the mutant. Hematoxylln and eosln, magnification X12.7.
usually two distinct bones which had not
fused at the caput. In some mes/mes mutants ribs may be asymmetrically fused as
a result of the abnormal xiphisternum.
The overall result is a wider thoracic basket in relation to the whole body. The skeletal preparations made from the two
known +/mes heterozygotes to determine
the effect of heterozygosity on skeletal
structure showed no abnormalities.
Skins of the mes/mes mutant were found
to be consistently wider and longer than
those of the normal +/? sibling, confirming the impression that the living mutant
seems to have excess skin (W = 8.6 ± 0.4
versus 6.0 ± 0.2; L = 11.3 ± 0.8 versus
10.8 ± 0.7; four pair of males). No significant differences in heart, liver, spleen, thymus, and adrenal weights were found in
the limited sample examined to date (n =
4 pairs). Peritoneal body fat was lacking in
all 11 homozygotes examined. In general,
mutants of both sexes weighed less than
their like-sex normal sibling controls until
Histology
The musculature of adult mutant mice was
dramatically increased in mass due to increased numbers of fibers rather than increased size of fibers. Both appendicular
and epaxial muscles were involved. The
muscular hyperplasia of mes/mes mice is
easily seen in cross sections of hip and
lumbar muscles (Figure 4). Muscle fiber
hyperplasia was more pronounced in male
than in female mutant mice, but was present in both sexes. Histologic examination
of achilles tendon of mutant mice revealed
that the collagen was mineralized, but neither bone nor cartilage was present in the
tendons. Other organs were without significant lesions. The sections of cerebrum
and other areas of the brain of mutants
had no lesions, which might have accounted for their unusual docile temperament.
Biochemical Body Composition
Analyses
Biochemical analyses of entire carcasses
showed that the mes/mes males (n = 4)
have a higher percent body water (67.8%
versus 63.4%), and lower percent body fat
(22.6% versus 29.6%) than their normal littermates. Differences in percent body protein (8.6% versus 6.0%) and mineral (0.99%
versus 0.86%) were not significant.
IGF-I and IGFBP Levels
The mean serum IGF-I level of mes/mes
mice (486 ± 34 ng/ml, n=7) did not differ
significantly from that of the +/? controls
(540 ± 35 ng/ml, n = 6). We found, however, that the total serum binding capacity
for IGF-I was reduced in mes/mes mice (n
= 7) compared to the +/? mice (n = 5),
and that the reduction was due primarily
to significant reduction (p = .003) in
IGFBP3 levels (IGFBP3 in mes/mes mice =
28.8 ± 3.1; in +/? = 54.8 ± 6.7).
Figure 5. Ventral view of 3 month old mes/mei male on the B6C3Fe-a/a hybrid background. Illustrating abnormal
testlcular position.
9 0 The Journal of Heredity 1996:87(2)
Reproduction
On the B6C3Fe genetic background both
sexes of mutant mice seldom breed. A few
homozygous females have become pregnant but failed to deliver viable offspring.
Male mutants are probably infertile because the testes are usually undescended.
Testes either remain in the abdominal cavity or come to rest outside the normal
scrotal position under the skin of the upper rear legs (Figure 5). In some cases the
Figure 6. Cross sections of testes from mes/mes mutant male and +/? age-matched llttermate. (a) tubule In abdominally located testis from mes/mes, note vacuolated cells
and no sperm In lumen; (b) tubule from opposite testis of the same mes/mes male as (a), but located under skin of leg, note less severely affected; (c) +/? llttermate control;
(d) tubule cross section from Xpl/Y male, testis located low In abdomen near Inguinal canal. All magnifications «• X1333
inguinal canal appeared blocked, while in
others the testes could be manually
pushed into the scrotal sac. Examination
of the inguinal area of one mes/mes mouse
revealed that the inguinal canal extended
through the inguinal ring halfway to the
location of the scrotal sac, if that were
present. Since no scrotum had developed,
the testes could descend into the inguinal
region along the inner aspect of the thigh.
The preputial glands and seminal vesicles
are approximately one-fourth the size of
control glands, suggesting that the mutant
mice are deficient in androgens. This
might account for the failure of the inguinal canal and scrotum to develop. Levels
of gonadal steroids have not been measured. Histologic examination of testis
sections showed that undescended testes
had fewer tubules with open lumens.
There were reduced numbers of mature
sperm and few sperm tails evident in the
lumens. The severity of the effect correlated with the position of the testis in the
abdominal cavity. One completely internalized testis was composed mostly of interstitial cells and had only type A spermatogonial cells, with multinucleate syncytia. Sertoli cells and cells with clumped
chromatin and vacuolated cytoplasm
(suggesting degeneration) often were
found in the center of the lumens (Figure
6a). Although testes positioned along the
leg or undescended testes with open inguinal canals had maturing germ cells
(Figure 6b), they appeared to be fewer in
number than in littermate controls (Figure
6c). The mouse mutations X-linked polydactyly (Xpl) (Sweet and Lane 1980) and
dominant hemimelia (DH) (Searle 1964)
also cause cryptorchidism, and a similar
histological phenotype was seen in undescended testes of Xpipi and Dh/+ mutant
males (Figure 6d). Testis weight is less in
mes/mes males than normal littermates,
consistent with the reduction in germ cell
numbers.
Genetic Analysis
Mesenchymal dysplasia is inherited as a
recessive mutation. The mating between
the original heterozygous +/mes male
(sire of the first mutant mice seen in the
Deviant Search) and the B6C3Fe-a/o F, female yielded all normal progeny. Matings
among these F, progeny resulted in the appearance of homozygous mes/mes affected animals in the F2 generation. Of 324 offspring from known carriers, 58 were classified as mes/mes, a frequency not significantly different from the expected
frequency of 0.25 (58/324 = 0.1790, x 2 =
3.2654, p > .07) for a recessive gene.
Tests for allelism were negative with
known mutations with one or more similar
skeletal defects, including brachymorphic
Sweet et aJ • Mesenchymal Dysplasia 91
Table 1. Two-point crosses between mes and genes on Chr 13
Phenotype of progeny
Cross
Female
1
2
mes +/+ bg
mes +/+ mu
3
mes mu/+ +
male
++
mes + + m* mes m Total
RE ± SE
X
X
mes +/+ bg
mes +/+ mu
130
241
50
63
98
5
57
248
396
X
mes mu/+ +
38
—
3
9
50
28.70 ± 5.75
17.3 upper 95%
confidence level
6.59 ± 3.65
• m — marker bg or mu.
(prri), brachypody (bp"), congenital hydrocephalus (c/?), flexed-tail (f), short-ear (se),
and extra toes (Xt1).
Linkage was first found with the recessive coat color mutation beige (bg) on
proximal Chr 13, suggesting that mes was
probably located toward the distal end of
Chr 13 (recombination = 28.74 ± 5.70)
(Table 1, cross 1). Subsequent crosses
with the more distal Chr 13 gene muted
(mu), a second recessive coat color mutation, confirmed this location. Since each
gene (mu, mes) is recognizable at birth, all
offspring were classified at birth and again
at weaning. No preweaning mortality and
no recombinants between mu and mes
(mice homozygous for both mutations)
were observed among the 396 F2 progeny
classified (Table 1, cross 2). The absence
of double mutants may have been due to
prenatal loss, although the observed litter
size suggested such loss was not substantial. If there was prenatal loss in cross 2 or
crosses 6 or 8, the loss does not appear
to affect the calculation of recombination
estimates, since these are internally consistent and consistent with previously
published data for these Chr 13 genes.
To determine if recombination had
taken place, 16 mu+/mu? females from the
F2 generation were progeny tested for mes
by mating to heterozygous +/mes males.
At least 12 offspring were raised from each
female tested. Five of 16 females tested
from this and subsequent crosses were
found to carry the mes gene, giving a recombination estimate of 18.52 ± 8.14% calculated by the method given in Deol and
Green (1966). A recombinant chromosome
with mu and mes in coupling was recovered and used for a coupling intercross
(Table 1, cross 3). To determine the exact
position of mes on Chr 13, mice carrying
mu and mes in coupling (from cross 3)
were mated to homozygous pearl (pe)
mice from the inbred C3HeB/FeJ-pe strain,
and F,s mated in the three-point intercross
shown in Table 2, cross 5. Homozygous
+pef?pe or mu+pe/mu ?pe progeny from
this cross were further tested by breeding
for the presence of mes. Seven of the 17
individuals tested were found to carry the
mes gene giving a recombination estimate
between pe and mes of 25.93 ± 9.49% [calculated according to the method given in
Deol and Green (1966)]. Two of the five mu
homozygotes from this cross carried a recombinant chromosome with mu and mes.
These data together with the data from
cross 2 above gave a total of seven mice
with recombination between mu and mes
in a total of 21 tested for a recombination
estimate of 20.00 + 7.41 (Deol and Green
1966). Further three-point intercrosses
(Table 2, crosses 6 and 7) were constructed by appropriate matings from such tested individuals.
Finally, a three-point intercross using a
fourth Chr 13 marker gene, Purkinje cell
degeneration (pcd), was done (Table 2,
cross 8). A female homozygote, pcd pelpcd
pe, obtained from a two point cross between B6C3Fe-a/a pcd X C3HeB/FeJ-pe F,s
(Table 3, cross 4), was used as a donor for
an ovarian transplant. The recipient female was then mated to a heterozygous
+/mes male from the B6C3Fe-a/a-mes colony. F, progeny of genotype +pcd pe/?+ + ,
were mated together to test for the presence of mes. F2 progeny from matings producing mes/mesoffspring were scored at
birth for mes, at 7 days for pe, and at
weaning for pcd. The cross between pcd
and pe shown in Table 3 was done to confirm that pcd maps to Chr 13.
Combining the data from all crosses for
each pair of loci gives the recombination
estimates shown in Tables 4 and 5. Although each of the three-point crosses involving the mes gene failed to produce
some of the expected progeny genotypes,
the data taken together allow us to position mes near the middle of Chr 13. Based
on the assumption that the least frequent
pairs of complimentary recombinant classes
is the double recombinant class we conclude that the most probable order is bg-mumes-pcd-pe (Figure 7). Cross 7 was omitted
from the calculations for the mu-pe interval
because the distance in this cross is significantly different from all other data for this
interval (cross 6; Cattanach et al. 1994; Lyon
and Meredith 1969). Dumpy (dpy), a mutant
gene with skeletal effects located on Chr 13
"fairly close" to mu (Hollander 1981) has
not been tested for allelism with mes and,
indeed, may be extinct. Wakasugi et al.
(1988) describe a mutation causing facial
and jaw abnormalities that was induced
by a transgenic insertion Into a Chr 13
gene. Although we did not test for allelism
with mes, no facial or jaw malformations
were seen in alizarin preparations of mes/
mes mutant mice.
Discussion
We have discovered and characterized a
new autosomal recessive mutation in the
mouse that causes malformations in many
mesoderm-derlved structures. Because of
the wide range of pleiotropy in affected
mutants, it is likely that the basic defect
occurs early in development. Thus, we
have named the mutation mesenchymal
dysplasia (mes). Some of the effects of mes
are common manifestations of pleiotropy
in mouse mutants of this type, including
polydactylism, tail kinks, and belly spots.
Skeletal defects, such as abnormalities of
the xiphistemum, may be common but difficult to detect in live animals. Located on
mouse Chr 13 are several somewhat simi-
Table 2. Three-point crosses confirming linkage of mes with genes on Chr 13
Cross
Female
x
mu ma +/++ pe
mu + pe/+ mes +
mu mes pe/+ + +
mes ++/+ pcd pe
9 2 The Journal oi Heredity 199687(2)
male
Phenotype of progeny
mu mes + + + pe
mu + pe
mu mes +/++ pe
mu + pe/+ mes +
145
119
150
22
+++
mes + +
21
2
28
7
mes pcd pe
mu mes
mes ++/+ pcd pe
66
49
37
+ pcd pe
32
+ mes +
32
77
20
+ pcd +
mu mes pe mu ++
11
39
43
mes + pe
+ mes pe Total
1
3
mes pcd + + + pe
261
301
223
Total
137
Table 5. Summary of all linkage data including
combined data from all crosses involving the
same genes
Table 3. Two-point cross between pcd and pe on Chr 13
Phenotype of progeny
Cross
Female
X
male
+ +
pcd +
+pe
pcdpe
Total
RE±SE
4
pcd +/+ pe
X
pcd +/+ pe
194
75
83
1
353
19.19 ± 3.99
lar pleiotropic mutations, including congenital hydrocephalus (ch), dumpy (dpy),
flexed-tail (f), and the extra toes mutations
in the GLHCruppel family member GLJ3
gene (GUV, GU3™, GliS*"*"). The effect of
mes on the formation of the sternum is
similar to that produced by the mouse mutations short ear (se) on Chr 9, ch, and Xt
on Chr 13 (Johnson 1986). Of the Chr 13
mutations only dpy maps in the region of
mes and we have been unable to test the
two genes for allelism. For a brief description of these genes see Green (1989), and
for a more complete analysis see Gruneberg (1952) or Johnson (1986). Of these
mutations only Xt is thought to be homologous to a human condition (Greig cephalopolysyndactyly syndrome).
The features unique to the mes mutation
are the wide spaced eyes, the loose skin,
the extremely thick tough footpads, and
docility of the homozygote. The excess
skin, wide face, hyperplasia of muscle,
mineralization foci in tendons, and abnormal sternum structure suggest overproduction of a growth factor or growth factor-related product that affects mesodermderived structures.
We have previously reported two other
mutations that cause excess growth of mesoderm-derived tissues, but mes produces
a phenotype distinctly different from either. Tight-skin (Tsk) causes excessive
subcutaneous connective tissue that is
caused by an increase in both cellularity
and amount of intercellular material in the
skin, and excess bone growth (Green et al.
1976). Tsk causes a defect in regulation of
biosynthesis of three specific collagen
molecules in dermal fibroblasts (Jimenez
et al. 1986). The recessive mutation progressive ankylosis (ank) causes excess
amounts of fibrous tissue, cartilage, and
bone primarily around the joints. Like Tsk,
ank causes an increase in cellular proliferation (Sweet and Green 1981). Excess
tissue in the mes/mes mutant is primarily
in the entire skin and in the muscle tissue
and is due to hyperplasia rather than hypertrophy.
Skeletal muscle hyperplasia is a striking
pathologic feature of mes/mes mutants. It
is similar to the bovine model of increased
musculature, double-muscling (DM) (Ashmore 1974). The observations of increased musculature of adult mutants and
the lack of peritoneal body fat led us to do
body composition analyses of the mutant.
Biochemical analyses, however, revealed
that although body water was increased
and body fat was reduced in mes/mes
males, body protein and mineral were not
significantly different from those of normal
littermates. The lack of statistically significant differences in whole body composition may be because the increased muscle
in mes/mes mutants is concentrated in
shoulder and hip areas and is not uniform
throughout the entire body. This is consistent with their reduced or unaffected organ weights, illustrating selectively increased protein accretion. The increased
musculature is probably not androgen
driven. If this were the case one would ex-
Interval
Crosses
RE + SE
mej-og
mes-mu
mes-pcd
mes-pe
mu-pe
pcd-pe
1
2,-3 ,5 , 6 , - 7
8
5,6, 7, 8
5,6
4,8
28.7 ± 5.8
17.2 ± 1.8
S29.1
12.7 ± 2.1
33.70 ± 2.8
15.4 ± 25
Data were combined using the average weighted method of Mather (1946).
• Recombination estimate from tested mice in crosses 2
and 6 used to calculate combined values.
pect androgen sensitive organs (salivary
glands, testis, and kidneys) to be enlarged,
whereas the mes/mes males have reduced
salivary glands, testes, and kidney mass.
Serum androgen measurements will be
needed to confirm this observation.
Since IGF-I has been shown to induce increased muscle protein synthesis (Pell
and Bates 1992; Tomas et al. 1991), we
considered the IGF-I/IGFBP axis as a possible mechanism for the increase in muscle protein accretion in mes/mes mice.
IGFBP3 is the largest of the IGFBPs and
serves to both transport IGF-I in circulation and to prolong its half-life (Baxter and
Martin 1989). While IGFBP3 in mes/mes
mice was reduced to approximately half
that of control mice, serum levels of IGF-I
in mes/mes mice were not different. An increase in IGF-I synthesis could account for
normal serum levels of IGF-I despite the
decreased serum IGFBP3 and potentiate
the biological action of IGF-I in skeletal
muscle tissue by allowing greater quantities of this growth factor to reach its target tissue. To establish the role of IGF-I/
IGFBP3 in this mutant, we plan further
studies to measure IGF-I and IGFBP3 synthetic rates, and to examine the response
T
Table 4. Recombination estimates for three-point crosses in Table 2 calculated by combining like
crosses
F, genotype
Linkage with mes:
mes +/+ mu
mes mu/+ +
mes +/+ pcd
mes +/+ pe
mes pe/+ +
Linkage among
other loci:
mu +/+ pe
mu pe/+ +
Crosses
mes +
+ nr
mes m
Total
RE ± SE
13.07 upper 95% confidence level
17.07 ± 1.82
29.1 upper 95% confidence level
12.95 ±3.71
12.59 ± 2.40
28.7 ±5.8
2,6
3,5,7
8
5,6,8
7
397
382
74
389
150
135
55
22
152
20
165
23
41
156
7
74
—
2
46
697
534
137
699
223
mupel+ +
5
6
7
++
177
196
170
++
pcdpe/+ +
8
87
mu +
33
39
0
pcd +
9
+ pe
49
37
3
+ pe
9
mu pe
2
29
50
pcd pe
32
Total
261
301
223
Total
137
• m — marker.
17.2 ±1.8
33.7 ± 2 £
12.7 ± 2 .
15,4 ±15
38.29 ± 5.21
31.84 ± 3.37
1.39 + 0.79
13.03 + 3.12
Figure 7. Map of Chr 13 summarizing all mapping
data In this article.
Sweet et al • MesenctiymaJ Dysplasia 9 3
of both proteins to regulatory stimuli such
as diet and exercise.
Reproductive failure in mes/mes males
appears to be secondary to failure of the
testes to descend normally, which may be
due to occlusion of the inguinal canal by
excess muscle. This conclusion is based
on two observations. First, abnormalities
in testis architecture and spermatogenesis, including cellular depletion, resemble
those seen in cryptorchid testes of Dh/+
and Xpl/Y males (this article), XXY mice
(Huckins et al. 1981), and other mammalian species rendered surgically cryptorchid (rhesus macaques, Resko et al. 1980;
opossum, Scott et al. 1979; and rats, Jones
et al. 1977) and in ipsilateral (right-sided)
cryptorchidism in man (MIM #219050;
GDB). Second, the degree of severity of
the testicular abnormalities was related to
the severity of cryptorchidism rather than
to the genotype.
Although the condition produced by the
mes mutation does not precisely resemble
a specific human syndrome, it has similarities with several. Of particular interest
are genes that are located in the proximal
region of the long arm of human Chr 5
(5qcen-q21) that is homologous to the region of mouse Chr 13 to which the mes
gene maps (GDB 1994). Contractual arachnodactyly (CCA), which is a mutation in
the fibrillin locus (FBN2) on human Chr
5q, has mesenchyme-derived tissue defects, including joint and skin laxity. This
locus maps to human Chr 5q23-q31 at the
distal end of the region homologous to
mouse Chr 13; however, it has recently
been mapped to Chr 2 in the mouse. Several spinal muscular atrophies also map to
proximal 5q in the Chr 13 homologous segment. Four other human conditions that
map to human Chr 5q have one or more
of the features of mes/mes mutants: camptomelic dwarfism (CMD1), Treacher-Collins Syndrome, which is associated with
deficient mesenchyme and facial dysmorphism, diastrophic dysplasia (DTD),
and limb girdle muscular dystrophy
(LGMD), which causes muscle weakness
in the shoulder and hip girdles (GDB
1994). All four of these diseases, however,
map to distal Chr 5q in the region homologous to mouse Chr 18.
Loci that map in the mes region of Chr
13 and are potential candidates for the
mes locus include Bmp6 (bone morphogenic protein; formerly Vgrl, Vg-1 related
protein), Fgfr4 (fibroblast growth factor receptor 4), Rasa (ras p21 GTPase activating
protein, also known as GAP), and Gasl
(growth arrest specific 1). Bmp6 is a ho-
9 4 The Journal of Heredity 1996.87(2)
mologue of Vg-1 in Drosophila and Xenopus and is a member of the transforming
growth factor beta gene superfamily. It is
involved in mesoderm initiation during
embryogenesis (Lyons et al. 1989). Fgfr4
encodes one of the high affinity receptors
for fibroblast growth factors, which appear to play an active role in mesoderm
induction and in growth and differentiation of mesodermal and neuroectodermal
cells (Givol and Yayon 1992). Although
Bmp6 and Fgfr4 are the most promising
candidates for mes, the present map position of mes is 10 to 15 cM distal to the
locations of these two genes (Avraham et
al. 1994; Dickinson et al. 1990). Rasa encodes a membrane protein that is associated with control of cell proliferation
through regulation of normal ras p21 protein (Trahey and McCormick 1987). Gasl
is one of several genes expressed at the G,,
growth arrest phase of mammalian cells
and is postulated to play a role in maintaining growth arrest (Schneider et al.
1988). A mutation in any of these genes
might lead to uncontrolled growth in one
or more tissues. More precise mapping of
mes is in progress to enable us to test candidate loci.
Mesenchymal dysplasia provides a new
mouse model for studying connective tissue defects. Its pleiotropic effect on a variety of mesenchyme-derived tissues suggests mes may be a mutation in a gene involved in early mesenchyme formation or
growth. Thus, determining the basic defect in mes/mes mutants should contribute
to understanding early mesenchyme development.
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Corresponding Editor Christine Kozak
Sweet et al • Mesenchymal Dysplasia 9 5