Cartilage anomaly (can); a new mutant gene in the mouse

By D. R. JOHNSON 0 1 JEAN M. WISE 0 1 0 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 1 From the M.R.C. Experimental Genetics Unit, Department of Animal Genetics, University College London 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. 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. - INTRODUCTION Normal can/can 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 canIcan 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 canIcan 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 canjean) 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. 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. 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. 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, 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. 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). 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 chondro (...truncated)