/. Embryol. exp. Morph. Vol. 43, pp. 71-84, 1978
Printed in Great Britain © Company of Biologists Limited 1978
71
Cartilage matrix deficiency (cmd) : a new autosomal
recessive lethal mutation in the mouse
By E. RITTENHOUSE, L. C. DUNN, J. COOKINGHAM,
C. CALO, M. SPIEGELMAN, G. B. DOOHER AND D. BENNETT 1
From the Memorial Sloan-Kettering Cancer Center, New York
SUMMARY
A new autosomal recessive lethal mutation in the mouse designated cartilage matrix
deficiency (cmd) is described. Homozygotes are dwarfed, and have abnormally short trunk,
limbs, tail and snout, as well as a protruding tongue and cleft palate. The abdomen is distended because the foreshortened rib cage and spinal column forces the liver ventrad from
its normal location. Histological and electron microscopic study reveals a deficiency of
cartilage matrix in tracheal cartilage and in all cartilagenous bones examined. The syndrome
closely resembles the rare lethal condition achondrogenesis, found in human infants, which
is also believed to be due to an autosomal recessive gene.
INTRODUCTION
A variety of different genetic syndromes that include disproportionate
dwarfism are known in mammals. We report here the occurrence in the house
mouse of a new autosomal recessive lethal mutation, cartilage matrix deficiency
(cmd), which closely resembles the human genetic disorder achondrogenesis. In
cmdjcmd animals the trunk, limbs, and tail are all abnormally short, the snout
is short and blunt with cleft palate and protruding tongue, and the abdomen
bulges noticeably. Death occurs just after birth because of breathing failure.
GENETICS
The first dwarfs observed were offspring of a brother-sister mating in a stock
in which unrelated mutations (T, Low, tf) were being maintained. Subsequent
matings of parents and relatives of the cmd offspring provided clear evidence
of the simple autosomal recessive inheritance of the mutant. Eighty-seven litters
each containing at least one cmd homozygote were observed within 12 h of
birth; in a total of 733 offspring, 180 (24-6%) were homozygous for cmd.
Dead offspring partially eaten by the mother, but recognizable as cmd homozygotes, were often found.
Eleven litters from proven +1 cmd x + jcmd matings were obtained by dis1
Authors' address: Section 6153, Sloan-Kettering Institute for Cancer Research, 425
East 68th Street, New York 10021, U.S.A.
72
E. RITTENHOUSE AND OTHERS
section just before birth. Of the 109 living embryos recovered, 81 were normal
and 28 (25-7%) were cmd homozygotes. This is the expected ratio if homozygous and has full penetrance and normal viability up to birth.
Live offspring from several matings of proven heterozygotes were classified
as 16 + / + and 29 +lcmdby progeny tests. The 1:2 ratio is evidence of normal
segregation and transmission of the lethal mutation. We have not attempted to
map cmd, but it shows no linkage with the chromosome 17 markers T and tf.
The cm«'mutant has been tested for allelism with the similar chondrodysplasia
(cho) mutant described by Seegmiller, Fraser & Sheldon (1971). Five litters
from matings between proven +\cmd and proven +jcho animals (kindly
provided by Professor R. E. Seegmiller) were dissected in late gestation. Of 44
embryos recovered, 43 were normal. The other was abnormally small (0-64 g
in a litter of mean weight 1-12 g) but was otherwise normal. Seventy-two offspring of the same parentage were observed at birth and no abnormalities
were noted in these. We conclude that cmd and cho are not allelic.
MORPHOLOGICAL OBSERVATIONS
Materials and Methods. All tissue for light or electron microscopy was taken
from embryos dissected on the 17th or 18th day after a vaginal plug indicated
that the mother had mated. Normal gestation is 19-20 days.
1. Paraffin sections. Tracheae (including larynx), livers and hindlimbs of cmd
and normal litter-mates were fixed in 10% buffered formalin (pH 7-2) or
Zenker's fixative, some limbs being decalcified with 0-5% nitric acid after
fixation. Paraffin sections were stained with the following materials: hematoxylin and eosin, toluidine blue in 30 % ethanol, periodic acid-Schiff technique
(PAS) for polysaccharides, or Van Gieson's picrofuchsin stain for collagen.
2. Frozen sections. Livers and mid-dorsal skin of cmd and normal littermates were frozen unfixed, sectioned in a cryostat and stained with Sudan
Black B in propylene glycol. The unfixed and alcohol-fixed (absolute ethanol
or methanol) sections of skin and liver were also stained with hematoxylin
and eosin, toluidine blue in 30 % alcohol, PAS, or Van Gieson's picrofuchsin.
Additional sections of liver were fixed in acetone-tetrahydrofuran and stained
with toluidine blue according to the procedure of Haust & Landig (1961) for
retaining highly soluble acid mucopolysaccharides.
3. Plastic sections. Tracheae of cmd and normal litter-mates were fixed in
phosphate-buffered (pH 7-4) 2 % glutaraldehyde with traces of CaCl2 (Spiegelman & Bennett, 1973) and postfixed in veronal-buffered (pH 7-4) 1 % Os0 4 , or
in the Os0 4 fixative only, dehydrated in ethanol and propylene oxide, and
embedded in Epon. Pieces of liver from cmd and normal litter-mates were
fixed in phosphate-buffered (pH 7-2) 3 % glutaraldehyde and subsequently in
phosphate-buffered (pH 7-4) 1 % Os0 4 , or in the Os0 4 fixative only, dehydrated,
and embedded in Maraglas.
Cartilage matrix deficiency (cmd) in mouse
73
Fig. 1. Newborn litter-mates homozygous for cmd. Both individuals show abnormally
short snouts and protruberant abdomens. The trunk and limbs are markedly
shorter than normal. The protruding tongue, typical of homozygotes, is visible
in the individual on the left.
Sections for electron microscopy were stained with aqueous uranyl acetate
followed by lead citrate and examined in a Philips 200 electron microscope.
Sections for light microscopy were stained with 1 % toluidine blue in 1 %
borax or with Richardson's methylene blue-azure A.
4. Skeletons. Newborns werefixedin 10 % formalin, skinned and eviscerated,
macerated in 2% KOH, stained with 0-1% alizarin red in 2% KOH, and
cleared in glycerol.
Gross characteristics. Animals homozygous for cmd are distinguished by
their short trunk and extremities, as well as their short snout, protruding
tongue and cleft palate (Fig. 1). The abdomen is strikingly protruberant, and
the liver appears enlarged. Observation at birth shows that cmd homozygotes
are born alive; they make a few gasping attempts to breathe but fail to do so
and die. The failure to breathe, confirmed by the fact that their lungs sink when
74
E. RITTENHOUSE AND OTHERS
Table l. Skeletal measurements of newborn cmd/cmd animals
and normal litter-mates
I. cmdlcmd newborn weighing 1-27 g and normal litter-mate, 1-38 g ; II. cmdjcmd
newborn weighing 1-04 g and normal litter-mate, 1-14 g. All measurements are in
mm.
cmdjcmd
II
Skull
Greatest length: top of
supra-occipital to tip of
premaxillary
Greatest width : across
parietals
Nasal length
Vertebrae
Atlas to last caudal
Last thoracic plus
nine posterior
Foreleg
Humerus
Radius
Ulna
Clavicle
Scapula
Hind leg
Femur
Tibia
Fibula
Foreleg
Humerus
Radius
Ulna
Clavicle
Scapula
Hindleg
Femur
Tibia
Fibula
Foreleg
Humerus
Radius
Ulna
Clavicle
Scapula
Hindleg
Femur
Tibia
Fibula
Mutant/
normal
Normal
II
10-2
9-7
0-88
101
7-0
1-4
6-8
1-5
6-9
1-9
6-7
1-8
15-4
151
21-4
20-7
50
50
7-9
Total length (cartilage bone)
7-5
20
1-7
2-1
2-0
1-6
2-1
4-4
3-7
4-7
4-4
3-6
4-6
0-45
0-45
0-45
1-3
1-4
3-9
3-6
0-36
40
40
40
40
3-9
40
0-49
0-42
0-40
2-8
2-4
30
2-8
2-6
2-6
2-1
2-8
2-7
2-4
0-39
0-42
045
0-85
0-32
2-0
2-6
2-5
2-0
2-4
2-4
0-40
0-38
0-41
20
1-6
1-6
11
0-9
1-3
2-4
0-7
0-7
0-9
10
1-9
1-7
1-6
Calcified length
10
10
1-3
2-3
0-9
0-9
10
10
Midshaft width
0-78
0-72
0-65
0-5
0-4
0-4
0-5
0-3
0-4
0-6
0-4
0-4
0-6
0-3
0-4
0-83
100
1-00
0-6
0-5
0-2
0-6
0-6
0-2
0-5
0-5
0-3
0-5
0-5
0-2
1-20
110
0-80
Cartilage matrix deficiency (cmd) in mouse
75
Fig. 2. A 1 /tm thick plastic section through tracheal cartilage from a normal 17to 18-day fetus. Chondrocytes are well-spaced and embedded in an abundant, lightly
staining matrix. Glutaraldehyde and osmium tetroxide fixation. Epon embedded.
Richardson's stain, x 900.
placed in water, probably results from a combination of factors including cleft
palate, protruding tongue and abdominal compression, as well as a defective
trachea and rib cage.
Skeletons. As seen in Table 1, cmd affects the appendicular skeleton more
severely than the axial skeleton or the skull. The long bones are reduced to less
than half normal length, while the spinal column is shortened by about 25%.
An exception is the clavicle which, significantly, is the only membrane bone of
the appendicular skeleton. Calcification appears to proceed normally in the
dwarfs, since the calcified regions of their long bones constitute essentially the
same proportion of the total cartilage bone length as in normals.
Thoracic, lumbar and sacral vertebrae of normal newborns contain three
clearly defined regions of calcification : one in the vertebral body and one in
each half of the vertebral arch. In cmd newborns, however, these three centers
are joined, except in the most posterior calcified vertebrae.
Cartilage. The most dramatic differences between cmd and normal morphology are found in the histology of cartilage.
76
E. RITTENHOUSE AND OTHERS
Fig. 3. A 1 fim thick plastic section through tracheal cartilage from a 17- to 18-day
fetus homozygous for cmd. Notice that chondrocytes appear crowded together
with very little intervening matrix compared with the normal. Numerous pyncotic
cells showing large, pale staining vacuoles and densely staining inclusions are
visible. Glutaraldehyde and osmium tetroxide fixation. Epon embedded. Richardson's stain, x 900.
Tracheal cartilage is especially important to the interpretation of this mutation because the tracheal rings are permanent hyaline cartilage, and thus present
no problems in distinguishing primary cartilage abnormalities from abnormalities that might be secondary to abnormal ossification. Sections of cmd and
normal tracheal cartilage in plastic sections show striking differences (Figs. 2, 3).
Normal cartilage contains well-spaced chondrocytes and a high proportion of
matrix (Fig. 2). Cmd tracheal sections show tightly packed chondrocytes, very
little matrix, and many pycnotic cells, which often contain one or more conspicuous vacuoles (Fig. 3). The matrix of both cmd and normal cartilage stains
with toluidine blue, but only the cmd matrix picks up picrofuchsin stain for
collagen. In fact, the cmd matrix is more clearly defined by picrofuchsin stain
than by toluidine blue. We must conclude that the abnormal matrix contains
unusual amounts or forms of collagen fibers.
Examination by electron microscopy of the normal and cmd tracheal cartilage
Cartilage matrix deficiency (cmd) in mouse
11
Figs. 4 and 5. Electron micrographs of chondrocytes and matrix from the tracheal
cartilage of a normal 17- to 18-day fetus. Osmium tetroxide fixation. Epon embedded.
Uranyl acetate and lead citrate stain.
Fig. 4. Chondrocytes contain abundant endoplasmic reticulum (arrow) and typically
show numerous surface projections. The matrix consists of abundant mucopolysaccharide-rich ground substance traversed by a loose feltwork of randomly
disposed collagen fibers. Notice that adjacent to the chondrocytes collagen fibers
are less abundant than elsewhere in the matrix, with the result that each cell is
surrounded by a pale halo, x 9500.
matrix supports this conclusion. In thin sections of normal tracheal cartilage,
chondrocytes are separated by broad avenues of matrix, a loose feltwork of
fine collagen fibers distributed in an amorphous ground substance (Fig. 4).
The area immediately surrounding each chondrocyte, however, has relatively
few collagen fibers. The cells are round to oval in shape and show numerous
short, tapered surface projections (Fig. 4). The cytoplasm is rich in ribosomes
and rough endoplasmic reticulum. The thin collagen fibers are randomly
arranged within the matrix (Fig. 5). Also scattered among the collagen fibrils
are numerous small, electron-dense granules, often with spikey profiles, interpreted as proteoglycans (Anderson & Sajdera, 1971 ; Pennypacker & Goetinck,
1976). This is the usual pattern characteristic of hyaline cartilage (Matukas,
Panner & Orbison, 1967).
6
EMB 43
78
E. RITTENHOUSE AND OTHERS
Fig. 5. Delicate, widely spaced unhanded fibers of collagen traverse the pale staining
matrix in all directions; irregularly shaped, small dense granules, believed to represent acid mucopolysaccharides, are scattered throughout the matiix. A few profiles
of portions of chondrocytes are included in the section, x 57000.
The matrix of cmd cartilage, on the other hand, is densely criss-crossed with
thick collagen fibers; the background of acid mucopolysaccharides is correspondingly sparse (Fig. 6). Furthermore, chondrocytes of cmd cartilage are
sometimes partly or wholly surrounded by a ring of collagenfibers(Figs. 6, 7).
Within this investment the collagen fibers are more closely packed than within
the rest of the matrix and often show a more uniform orientation (Fig. 7).
Unlike normal chondrocytes, these cells in the mutant have few cytoplasmic
processes which are usually short and blunt (Fig. 6). Not only within the fibrous
sheath, but everywhere within the matrix the collagen fibers are straighter,
longer and thicker than those found in normal matrix (Figs. 6, 7). The granules
in the matrix of the mutant (Fig. 8) frequently occur in clusters, and are considerably larger than in normal matrix, less dense, and frequently round or
amorphous in shape (compare Fig. 8 with Fig. 5).
Sections of normal cmd leg cartilage were also studied. Sections of normal
embryonic legs show the usual epiphyseal cartilage zones (Fig. 9). In cmd
epiphyseal cartilage, cells near the marrow cavity show hypertrophic changes,
but there is no indication of column formation, and chondrocytes are grouped
in cell nests without consistent orientation (Fig. 10). The cmd chondrocytes
are so closely packed that matrix appears to be lacking, but decalcified sections
Cartilage matrix deficiency (cmd) in mouse
79
Figs. 6-8. Electron micrographs of developing cartilage from a 17- to 18-day fetus
homozygous for cmd. Osmium tetroxide fixation. Epon embedded. Uranyl acetate
and lead citrate stain.
Fig. 6. Chondrocytes resemble those from normal individuals in containing abundant
rough endoplasmic reticulum (arrow). In the mutant, however, cells exhibit fewer
and blunter surface projections and often contain lipid inclusions (L). The mucopolysaccharide matrix is very sparse and filled with irregularly arranged collagen
fibers. A dense collagenous investment encloses one of the chondrocytes in the
field, a feature not observed in normal cartilage at the same stage of development.
The whorl of concentric membranes, also ensheathed by densely packed collagen
fibers, is probably a pycnotic chondrocyte (C). x 9500.
stained with toluidine blue show that metachromatic matrix does exist. It is
difficult to compare the intensity of toluidine blue metachromasia between cmd
and normal matrix because the normal matrix is so much more open. Here
again, cmd matrix stains with the picrofuchsin stain for collagen whereas the
normal matrix does not.
Liver and skin. Because the livers of cmd newborns are so obviously enlarged,
it was first thought that the mutation might be a mucopolysaccharide storage
disease resembling Hurler's syndrome in man (Dunn & Bennett, 1970). Histological, histochemical and electron microscopic study, however, revealed no
abnormality of the liver except congestion with blood, severe in newborns and
6-2
80
E. RITTENHOUSE AND OTHERS
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Fig. 7. In this micrograph the orderly arrangement of thick collagen fibers within
the sheath can be compared to the random arrangement within the matrix generally.
Notice that the majority of collagen fibers in the matrix from the mutant are
thicker and straighter than those from normal individuals, x 28 500.
slight or moderate in embryos. This is consistent with the observation that
cmd livers are darker than normal and seems a sufhcient explanation for the
enlargement.
Cryostat sections of mid-dorsal skin and paraffin sections of the whole legs
indicate no abnormal distribution of collagen or storage of other substances in
cmd skin.
DISCUSSION
The recessive mutation cartilage matrix deficiency (cmd) produces a dwarfism
in mice caused by severely defective cartilage matrix. This deficiency results
in a striking reduction in the size of the cartilage bones. The dimensions of
Cartilage matrix deficiency (cmd) in mouse
81
Fig. 8. In this micrograph of cmd cartilage the matrix is seen to be studded with
amorphous granules frequently occurring as small aggregates. These granules may
represent acid mucopolysaccharides, but they are larger and less dense than granules
seen in normal matrix (Fig. 5). The densely packed, long, thick collagen fibers of
the mutant cartilage can also be compared to the thin fibers of the normal cartilage,
x 57000.
membrane bones are also reduced, but to a lesser degree. This can be plausibly
explained as a conformity of developing membrane bones to dimensions determined by the cartilage bones. We found no evidence of direct effect on other
tissue.
The matrix surrounding each chondrocyte in cmdjcmd individuals is greatly
reduced in area and the fine structure of the matrix is altered. Electron micrographs of tracheal cartilage sections show abnormally heavy collagen deposits
in the matrix, as well as an altered form and distribution of acid mucopolysaccharides. The collagen fibers of the mutant matrix are abnormally large and
closely packed. Chondrocytes are also closely packed and often show signs of
degeneration. Perhaps the altered mucopolysaccharide ground substance permits the greater degree of aggregation and cross-linking of collagen in the
mutant than in the normal cartilage.
The possible influence of the close investment of collagenous fibers upon the
chondrocytes in the mutant is unknown. Chondrocytes from cmd homozygotes
typically have shorter and blunter surface projections than their normal counterparts. Perhaps the closely apposed collagenous sheath restricts the mobility of
82
E. RITTENHOUSE AND OTHERS
Fig. 9. Light micrograph of a paraffin-embedded epiphyseal plate from the tibia of
a normal 17- to 18-day fetus showing typical zonation due to the gradual transition
of chrondrocytes from flattened, 'resting' cells to round hypertrophied cells as they
approach the developing marrow cavity. The zone of calcification of the cartilage
matrix and of bone deposition is densely staining. The arrangement of chondrocytes
into longitudinally oriented columns is apparent between the zone of resting cells and
the zones of hypertrophy and calcification. Formalin fixation. Hematoxylin and
eosin stain, x 250.
Fig. 10. Comparable section to Fig. 9 from a mutant of the same age. Chondrocytes
are very closely packed due to the scanty extracellular matrix. No clear-cut arrangement of chondrocytes into longitudinal columns is evident although the cells show
signs of hypertrophy adjacent to the developing marrow cavity. Formalin fixation.
Hematoxylin and eosin stain, x 250.
Cartilage matrix deficiency (cmd) in mouse
83
the plasma membrane. Other evidence has correlated the irregular shape of
normal chondrocytes of tracheal cartilage with the release of mucopolysaccharides (Seegmiller et at. 1971). Thus, the smoothly surfaced chondrocytes typical
of the mutant may reflect reduced release of mucopolysaccharides.
A number of mutations producing disproportionate dwarfism have been
described in the mouse. These are all recessive, and include: achondroplasia
(en) (Lane & Dickie, 1968); brachymorphic (bm) (Lane & Dickie, 1968);
cartilage anomaly (can) (Johnson & Wise, 1971); chondrodysplasia (cho)
(Seegmiller e£ #/. 1971); phocomelic (pc) (Gluecksohn-Waelsch, Hagedorin &
Siskin, 1956, Siskin & Gluecksohn-Waelsch, 1959); short head (sho) (Fitch,
1961); stubby (stb) (Lane & Dickie, 1968); and stumpy (stm) (Wallace, 1973).
Although all of these mutations share some phenotypic features of cmd, each
is sufficiently different to exclude identity. We have not, however, tested for
allelism except in the case of cho, which we know not to be allelic to cmd.
Cho homozygotes are superficially very similar to cmd/cmd animals, being
almost indistinguishable on gross inspection. However, unlike cmd animals,
cho homozygotes appear to have abnormalities in the number, size, shape, or
distribution of chondrocytes in the resting zone in long bones. The cartilage
matrix is abnormal, but again in a different way. The cho matrix has reduced
metachromasia, and unusually large collagen fibrils, which show the 64 nm
banding pattern that is not normally seen in cartilage matrix. Thus, in cho the
matrix seems to be normal in amount, although perhaps deficient in (particular)
acid mucopolysaccharides (Seegmiller et al. 1971), whereas in cmd there may
be an absolute deficiency or deviant distribution of matrix.
One of the mutations listed above (can) seems to produce effects very similar
to cmd, but not as severe. At birth, can/can animals are smaller than normal,
have a domed skull, short limbs, short body, and a bulging abdomen. They die
usually about 10 days postnatally of breathing difficulties. In the light microscope cartilage cells are seen to be crowded together, with about 50 % more
cells per area than in normals. In the electron microscope the intercellular
matrix is dense but contains apparently normal collagen, of the sort typical of
cartilage (Johnson & Wise, 1971).
In the human, the most prevalent form of disproportionate dwarfism is
achondroplasia, known to be due to a dominant gene (Rimoin et al. 1970;
Bailey, 1973). Cmd clearly does not resemble achondroplasia either in genetic
determination or in phenotypic detail. Thanatophoric dwarfism, of uncertain
genetic etiology, can also probably be differentiated from cmd since the trunk
is of normal length (Kaufman, Rimoin, McAlister & Kissane, 1970).
Another rare form of human dwarfism, the lethal condition achondrogenesis
appears to be similar to cartilage matrix deficiency in every detail. Achondrogenesis is probably due to an autosomal recessive. Affected individuals are
stillborn or die just after birth and show micromelia, a short trunk, and a
distended abdomen with engorged liver. Histological examination of long bones
84
E. RITTENHOUSE AND OTHERS
reveals very deficient cartilage matrix, and disorganized endochondral ossification (Saldino, 1971; Bailey, 1973).
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{Received 1 March 1977, revised 27 May 1977)