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.

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 cmd/cmd 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.

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 +/cmdx +/cmd matings were obtained by dissection 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 cmd has full penetrance and normal viability up to birth.

Live offspring from several matings of proven heterozygotes were classified as 16 + / + and 29 + /cmd by 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 cmd 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 + /cho 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 off spring of the same parentage were observed at birth and no abnormalities were noted in these. We conclude that cmd and cho are not allelic.

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 (Spiegel-man & Bennett, 1973) and postfixed in veronal-buffered (pH 7·4) 1 % OsO4, or in the OsO4 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 % OsO4, or in the OsO4 fixative only, dehydrated, and embedded in Maraglas.

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 were fixed in 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 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.

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.

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.

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.

Table 1.

Skeletal measurements of newborn cmd/cmd animals and normal litter-mates

Skeletal measurements of newborn cmd/cmd animals and normal litter-mates
Skeletal measurements of newborn cmd/cmd animals and normal litter-mates

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.

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.

Fig. 2.

A 1 μm thick plastic section through tracheal cartilage from a normal 17-to 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, ×; 900.

Fig. 2.

A 1 μm thick plastic section through tracheal cartilage from a normal 17-to 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, ×; 900.

Fig. 3.

A 1 μm 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, ×; 900.

Fig. 3.

A 1 μm 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, ×; 900.

Examination by electron microscopy of the normal and cmd tracheal cartilage 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).

Fig. 4.

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, ×; 9500.

Fig. 4.

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, ×; 9500.

Fig. 5.

Delicate, widely spaced unbanded 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 matrix. A few profiles of portions of chondrocytes are included in the section, ×;57000.

Fig. 5.

Delicate, widely spaced unbanded 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 matrix. A few profiles of portions of chondrocytes are included in the section, ×;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 collagen fibers (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).

Fig. 6.

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). ×; 9500.

Fig. 6.

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). ×; 9500.

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, ×;28 500.

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, ×;28 500.

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, ×;57000.

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, ×;57000.

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 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.

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, ×;250.

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, ×;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, ×; 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, ×; 250.

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 slight or moderate in embryos. This is consistent with the observation that cmd livers are darker than normal and seems a sufficient 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.

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 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 cmd/cmd 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 the plasma membrane. Other evidence has correlated the irregular shape of normal chondrocytes of tracheal cartilage with the release of mucopolysaccharides (Seegmiller et al. 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 (cn) (Lane & Dickie, 1968); brachymorphic (bm) (Lane & Dickie, 1968); cartilage anomaly (can) (Johnson & Wise, 1971); chondrodysplasia (cho) (Seegmiller et al. 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 reveals very deficient cartilage matrix, and disorganized endochondral ossification (Saldino, 1971; Bailey, 1973).

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