/. Embryo/, exp. Morph. Vol. 25, J,pp. 21-31, 1971
21
Printed in Great Britain
Cartilage anomaly (can); a new mutant
gene in the mouse
By D. R. JOHNSON 1 AND JEAN M. WISE 1
From the M.R.C. Experimental Genetics Unit, Department of
Animal Genetics, University College London
SUMMARY
Cartilage anomaly (symbol can) is a recessive gene in the mouse producing achondroplasia.
Abnormal mice die at about 10 days after birth. Light microscopy reveals a systemic deficiency in the cartilaginous matrix. Electron microscopy suggests that the collagen of the
matrix is normal and that the mucopolysaccharide component is reduced.
INTRODUCTION
Although the literature of achondroplasia is vast (Landauer's (1969) bibliography of micromelia lists 1755 references) our understanding of the factors
that control reduced cartilage formation is poor. The present paper gives a
preliminary description of a recessive gene, cartilage anomaly (can) in the mouse,
which produces achondroplasia, possibly due to a deficiency of mucopolysaccharides in the cartilaginous matrix.
ORIGIN AND GENETICS
The gene can arose in 1965 as a mutation in the deafness (dri) stock at the
M.R.C. Experimental Genetics Unit at U.C.L. It segregates as a single recessive
gene (Table 1). The fit to the expected 3:1 ratio is poor (xl = 5"62, P = 002)
with a deficit of abnormals. This can be accounted for by death of homozygotes
in utero. The + x + matings have a mean litter size of 5-65, which is 0-31 more
than +/canx +/can matings; in 82 litters this corresponds to 25-4 young.
Table 1. Segregation o/can
Offspring
Mating type
1.
2.
1
+ /can x + lean
+x +
Normal
can/can
Total
No. of
litters
Litter
size
350
260
88
438
260
82
46
5-34
5-65
Authors' address: M.R.C. Experimental Genetics Unit, Department of Animal Genetics,
University College London, Wolfson House, 4 Stephenson Way, London NW 1 2HE, U.K.
22
D. R. JOHNSON AND J. M. WISE
Assuming that these are all canlcan and adding them to the classified young, the
ratio becomes 350:103, which is not significantly different from 3:1 (xl = 1"2).
MATERIAL AND METHODS
Litters of embryos (from + jean x + jean matings) aged 15 days, 16 days and
at term were stained with methylene blue; also one can I can and a normal
litter-mate aged 1 day. Alizarin clearance preparations were made of pairs of
animals aged 11, 15, 17, 19 and 38 days.
Histological observations were made by traditional methods (formalin
fixation, 10 /A, sections stained in H and E or Mallory) on litters of embryos from
+ jean x + jean matings and on canlcan mice and their normal litter-mates.
In embryonic material (six unclassifiable embryos aged 14 days; two normal,
two can I can aged 17 days) the knee joints only were sectioned. In newborn
mice (two normal, two can/can) knee joints, larynx, trachea, calvarium and
ribs were examined. In 14-day-old mice (two normal, two can/can) the anterior
part of the head, eyelids, external ears, suprascapular processes and the last
centimetre of the tail were also sectioned.
For electron microscopy femoral heads were dissected from ten normal and
fourteen can/can newborn mice, fixed in glutaraldehyde and postfixed in osmium
tetroxide. Ossification was studied in segments of rib (ten normal, seven can/can)
and calvarium (five normal, six can jean) from newborn mice, and also in two
pairs of 14-day-old mice. The latter were decalcified in 1 % formic acid in
cacodylate-sucrose buffer after osmium postfixation. Ultrathin sections were
stained with lead citrate and uranyl acetate.
Oxygen saturation of whole haemolysed blood was determined by a modification of the method of Johnston (1963). Nine-day-old can/can mice and their
normal litter-mates were beheaded and 0-1 ml of blood collected in a 1 ml
disposable syringe, the dead-space of which had been filled with heparin
solution. The blood was rapidly transferred to spectrophotometer cells filled
with a solution of 15 % Triton-X 100 in 0-1 % Na2CO3 overlaid with butanol.
After gentle mixing the cells were placed in a Unicam SP 500 spectrophotometer
and optical density was read at 650 and 805 m/t. Fully oxygenated and reduced
standards were prepared by the methods of Johnston.
RESULTS
The canjean mice are smaller than their normal litter-mates at birth (34
normal mice weighed 1-334 ± 0-065 g; their 23 can/canslitter-mates 1-141 ± 0-062 g;
t = 5-46, P = 0-02). They gain weight slowly (Fig. 1). At 18 days the mean
weight of the abnormals is 2-7 g (normal litter-mates 5-4 g; abnormal/normal
(A/N) = 0-5). The longest surviving can/can weighed 4-29 g at 38 days (normal
litter-mate 16-09 g; A/N = 0-27). This was exceptional, for death usually occurs
at around 10 days.
Cartilage anomaly (can)
23
6r
3
I
4
I
5
I
6
I
7
i
8
I
I I
I I I
I
I I
9 10 11 12 13 14 15 16 17 18 19 20 21
Age (days)
Fig. 1. Growth curve of two can/can <$<$ and their four normal litter-mates. The abnormals in this litter were unusually long-lived: both died at 21 days. However, the
increase in weight until the day of death is typical.
Table 2. Measurements (in mm) of alizarin preparations of pairs of can/can mice
and normal litter-mates
15 days
days
A/N
N
Skull
14-4
length
Skull
8-3
width
Body 220
length
Tail
290
length
Femur 4-8
length
Femur 0-7
width
Tibia
7-0
length
Tibia
0-6
width
Middle 4-7
metatarsal
length
11-5 0-80
8-3
N
176
17 days
A/N
12-6 0-72
19 days
N
A
A/N
16-5
12-8 0-76
38 days
N
A
A/N
N
A
A/N
171
140
0-82
20-5
150
0-73
100
106
91
0-86
10-7
8-8
082
100
9-5
0-95
130
100
0-88
17-7 0-81
285
186
066
310
197
064
31-5
200
0-64
490
210
0-43
160
0-55
470
240
0-51
410
220
0-54
400
200
0-50
700
38-9
0-56
3-4 0-71
74
4-5 0-61
7-4
40
0-54
7-3
4-8
0-66
12-8
4-8
0-38
0-6
0-86
09
0-8
0-89
0-8
0-8
1-00
0-7
0-7
100
1-3
08
0-62
4-3
0-61
8-5
50
0-59
9-8
4-5
0-46
9-7
5-5
0-57
159
7-3
0-46
0-5
0-83
0-7
0-5
071
0-7
06
086
0-7
0-5
0-71
10
0-7
0-70
2-0 0-43
5-4
2-8
0-52
5-0
2-5
0-50
4-6
2-9 0-63
7-7
4-1
0-53
24
D. R. JOHNSON AND J. M. WISE
Homozygotes can be distinguished at birth by their shortened domed skulls
and short limbs as well as by their small size. These abnormalities can be
traced back with certainty to 17-day embryos; can/can mice have bulging
abdomens from birth onwards.
The bones of the can/can skeleton are shorter and thicker than those of their
normal litter-mates (Table 2; Fig. 2). There is malocclusion of the incisors,
5 mm
Fig. 2. Right forelimbs of (A) normal and (B) can/can litter-mates aged 11 days.
Camera lucida drawings of alizarin preparations.
Fig. 3. Lateral views of the thorax of a 15-day-old can/can (B) and a normal littermate (A). Cartilage is stippled.
Cartilage anomaly (can)
25
because the upper jaw is considerably shortened. The ribs are short and the
rib-cage flattened dorso-ventrally and hence reduced in volume (Fig. 3). There
is marked scoliosis of the thoracic vertebral column and ectopic calcification of
the neck muscles. The junction of osseous and cartilaginous rib has a spatulate
appearance. Ossification of the epiphysial plates is less than in normals of the
same age: this could be a reflexion of general retardation. The defects of the
osseous skeleton are preformed in cartilage. Moreover, methylene blue was not
taken up as strongly by mutant as by normal cartilage (Fig. 4).
2 mm
I
Fig. 4. Right forelimbs of (A) normal and (B) canlcan litter-mate. Fifteen-day-old
embryos. Camera lucida drawing of methylene-blue preparation. Dark areas
represent heavier staining.
The chondrocytes of the head of the femur of newborn can/can mice are
closely packed (Fig. 5) with less interstitial material than normal. Counts of
selected fields, using a graticule in the microscope eyepiece, showed about 50 %
more chondrocytes per unit area in the abnormal. The epiphysial plate is thin
and the proliferating cartilage columns poorly aligned. The zone of hypertrophic
cartilage is less than half the normal thickness with a scarcity of vacuolated
chondrocytes, and the line of calcification is irregular. No abnormality was seen
in the structure or ultrastructure of the bone formed.
Abnormal cartilage was well established in the 17-day embryonic knee joint,
with crowding of chondrocytes. No abnormal cartilage could be found in a litter
of six 14-day-old embryos from known heterozygous parents.
All cartilaginous regions from the 14-day-old can/can mice sectioned were
abnormal. The appearance was similar to that seen in the newborn knee.
Cartilaginous entities were smaller than in the normal litter-mate, with an
increased number of cells per unit area embedded in a sparse, poorly staining
matrix.
In electron micrographs the nucleus of the normal newborn chondrocyte has
prominent electron-dense areas which are absent in canlcan (Figs. 6, 7). Little
glycogen was observed in the cytoplasm of normal midzone chondrocytes, but
glycogen deposits increased markedly in the zone of hypertrophy. In contrast
can/can chondrocytes from all areas regularly showed polar deposits of darkly
D. R. JOHNSON AND J. M. WISE
Fig. 5. (A, B) Longitudinal sections through the head of the femur of a normal (A) and
a can/can (B) newborn mouse (H and E, x 100). (C, D) Appearance of selected
regions of normal (C) and can/can (D) newborn femoral cartilage (H and E, x 390).
Cartilage anomaly (can)
Fig. 6. Midzone chondrocyte from normal newborn mouse from the can stock. Note
heterochromatic nucleus.
27
28
D. R. JOHNSON AND J. M. WISE
'Mi
Fig. 7. Midzone chondrocyte from newborn can/can mouse. Note uniformity of
nucleus and deposits of glycogen (g) in cytoplasm.
Cartilage anomaly (can)
Fig. 8. High-power views of portions of cartilaginous matrix from normal (A) and
canjcan (B) newborn femur.
29
30
D. R. JOHNSON AND J. M. WISE
staining glycogen. The can/can intercellular matrix appears dense in low-power
micrographs. In high-power views (Fig. 8) well-formed collagen fibres can be
seen embedded in a reduced interfibrillar matrix.
DISCUSSION
Lane & Dickie (1968) reported three recessive genes (achondroplasia, en;
brachymorphic, bm; stubby, stb) causing disproportionate dwarfing in mice.
They comment on the similarities between these mutants, and indeed their
description fits the can/can mouse also: 'All skulls are shortened but not noticeably narrowed. All axial skeletons are shortened, and most severely in the tail.
All appendicular skeletons are shortened but none shows any obviously greater
reduction in the hind than in the forelimbs nor in the distal than in the proximal
bones.'
The magnitude of the effect seems greater in can/can than in cn/cn mice
(the most extreme of Lane & Dickie's trio) as can/can animals die early, while
some en/en survive to breed. It is difficult to account for the death of can/can
mice. They may die of complications arising from a crowded thorax and
abdomen: reduced nasal passages may also have an effect. Lane & Dickie
found cyanosis in enjen mice which survived weaning. No evidence of this has
been seen in can/can mice and blood oxygen levels are normal (Table 3).
Table 3. Percentage of HbO2 in blood of can/can and their normal
litter-mates
Age (days)
No.
HbO2 (%)
+ /+
9
3
85-28 ±0-71
can/can
9
3
84-93 ±0-90
Konyukhov & Paschin (1967), who performed reciprocal bone transplants,
concluded that reduced bone growth in en I en was due to primary gene action in
the chondrocytes of cartilage bone. It is evident from light micrographs that
the chondrocytes of can/can are separated by too little matrix, in cartilage bones
and in the visceral skeleton. Cartilaginous matrix consists of interlacing collagen
fibres in an amorphous material which is largely mucopolysaccharide. Electron
micrographs suggest that in can/can the mucopolysaccharide is the deficient
component, the collagen fibres seeming well organized. The reduced staining
of the matrix with methylene blue also supports this observation.
A similar but more extreme situation is found in mice injected with oestradiol
(Silberberg, Hasler & Silberberg, 1965). Injection of this steroid hormone leads
to changes in the chondrocytes which result in the formation of a matrix rich
in thick collagen fibres but with reduced mucopolysaccharides. Inside the cells
glycogen is accumulated. Here the similarity ends, however, as oestradiol leads
to marked hypertrophy of the Golgi apparatus and endoplasmic reticulum
Cartilage anomaly (can)
31
not seen in can. Silberberg et al. suggest that oestradiol may interfere with the
production of polysaccharides or their removal from the cell. In can it is
possible that decreased polysaccharide formation by the chondrocytes leads to
sparsity of mucopolysaccharides in the matrix and accumulation of glycogen
(a raw material for polysaccharide synthesis) in the cells.
Shepard, Fry & Moffett (1969) studied the articular cartilage of the newborn
achondroplastic rabbit (en/en). They found an increase in the number of
degenerating chondrocytes with increasing distance from the free surface of the
tissue, and illustrate degenerating cells from achondroplastic animals. Degenerating cells were not very common in our material, certainly not more
common in can/can than in their normal litter-mates.
The hypothesis of a deficient or changed mucopolysaccharide content in
canlcan cartilage is currently being investigated by other means, and will be
fully reported in due course. It may be that polysaccharides are abnormal
elsewhere in the can/can mouse and that this leads to the sudden demise of an
animal which, although small, is still gaining weight up to the time of death.
RESUME
Cartilage anomaly can; un gene mutant nouveau chez la souris
Cartilage anomaly (symbole can) est une gene recessif qui chez la souris produit de
J'achondroplasie. Les souris anormales meurent aux environs du lOe jour apres la naissance.
Le microscope photonique demontre une deficience systematique de la matrice cartilagineuse.
La microscopie electronique fait suggerer que le collagene de la matrice serait normal et
que le composant mucopolysaccharidique serait reduit.
REFERENCES
JOHNSTON, G. W. (1963). Oxygen saturation of blood. Stand. Meth. din. Chem. 4, 183-189.
KONYUKHOV, B. V. & PASCHIN, Y. V. (1967). Experimental study of the achondroplasia gene
effects in the mouse. Ada biol. hung. 18, 285-294.
W. (1969). A bibliography on micromelia. In Limb Development and Deformity:
Problems of Evaluation and Rehabilitation, pp. 540-621. Springfield: Charles C. Thomas.
LANE, P. W. & DICKIE, M. M. (1968). Three recessive mutations producing disproportionate
dwarfing in mice. /. Hered. 59, 300-308.
SHEPARD, T. H., FRY, L. R. & MOFFETT, B. C. (1969). Microscopic studies of achondroplastic
rabbit cartilage. Teratology 2, 13-22.
LANDAUER,
SILBERBERG, R., HASLER, M. & SILBERBERG, M. (1965). Submicroscopic response of articular
cartilage of mice treated with estrogenic hormone. Am. J. Path. 46, 289-305.
(Manuscript received 13 May 1970)