/. Embryol exp. Morph. Vol. 39, pp. 279-284, 1977 279 Printed in Great Britain Ultrastructural observations on stumpy (strri), a new chondrodystrophic mutant in the mouse By D. R. JOHNSON 1 From the Department of Anatomy, School of Medicine, University of Leeds SUMMARY Stumpy (stm) is a new chondrodystrophic mutant in the mouse. Light microscopy of carti- lage reveals a slightly increased mitotic rate, more chondrocytes than is usual per lacuna and a wide zone of hypertrophy. Electron microscopy shows that many chondrocytes are in close approximation with some tight junctions: in cartilage from 14-day-old mice there is much interdigitation and folding of the cell membranes of adjacent chondrocytes. INTRODUCTION In recent years there has been a renewal of interest in hereditary chondro- dystrophy in both man and laboratory animals (see Rimoin, 1975, for a review). This has been largely due to the introduction of biochemical and ultrastructural studies on abnormal cartilage. It is becoming increasingly apparent, as descrip- tions of genetically distinct conditions accrue, that the chondrodystrophic phenotype can be produced by any one of a number of clearly distinct anomalies reflecting different ultrastructural or biochemical parameters. Thus, the gene for nanomelia in the chick, and possibly chondrodystrophy (cho) in the mouse, affect acid mucopolysaccharide synthesis (Fraser & Goetinck, 1971; Seegmiller, Fraser & Sheldon, 1971; Seegmiller, Ferguson & Sheldon, 1972; Pennypacker & Goetinck, 1976), cartilage anomaly (can) in the mouse seems to affect collagen synthesis (Johnson & Hunt, 1974), brachymorphism affects sulphation of acid mucopolysaccharides (Orkin, Pratt & Gill, 1976), whilst the achondroplastic rabbit (ac) has a defect in mitochondrial oxidative phosphorylation (Bargman, Mackler & Shepard, 1972). Achondroplasia (en) in the mouse is unique in having no known clear ultrastructural or biochemical defect (Silberberg & Lesker, 1975; Silberberg, Hasler & Lesker, 1976). Stumpy is yet another genetically distinct chondrodystrophic dwarf with a distinctive cartilage phenotype. 1 Author's address: Department of Anatomy, School of Medicine, University of Leeds, Leeds LS2 9NL, U.K.
Transcript
/ . Embryol exp. Morph. Vol. 39, pp. 279-284, 1977 2 7 9Printed in Great Britain
Ultrastructural observations on stumpy (strri), anew chondrodystrophic mutant in the mouse
By D. R. JOHNSON1
From the Department of Anatomy, School of Medicine,University of Leeds
SUMMARY
Stumpy (stm) is a new chondrodystrophic mutant in the mouse. Light microscopy of carti-lage reveals a slightly increased mitotic rate, more chondrocytes than is usual per lacuna anda wide zone of hypertrophy. Electron microscopy shows that many chondrocytes are inclose approximation with some tight junctions: in cartilage from 14-day-old mice there ismuch interdigitation and folding of the cell membranes of adjacent chondrocytes.
INTRODUCTION
In recent years there has been a renewal of interest in hereditary chondro-dystrophy in both man and laboratory animals (see Rimoin, 1975, for a review).This has been largely due to the introduction of biochemical and ultrastructuralstudies on abnormal cartilage. It is becoming increasingly apparent, as descrip-tions of genetically distinct conditions accrue, that the chondrodystrophicphenotype can be produced by any one of a number of clearly distinct anomaliesreflecting different ultrastructural or biochemical parameters.
Thus, the gene for nanomelia in the chick, and possibly chondrodystrophy(cho) in the mouse, affect acid mucopolysaccharide synthesis (Fraser & Goetinck,1971; Seegmiller, Fraser & Sheldon, 1971; Seegmiller, Ferguson & Sheldon,1972; Pennypacker & Goetinck, 1976), cartilage anomaly (can) in the mouseseems to affect collagen synthesis (Johnson & Hunt, 1974), brachymorphismaffects sulphation of acid mucopolysaccharides (Orkin, Pratt & Gill, 1976),whilst the achondroplastic rabbit (ac) has a defect in mitochondrial oxidativephosphorylation (Bargman, Mackler & Shepard, 1972). Achondroplasia (en) inthe mouse is unique in having no known clear ultrastructural or biochemicaldefect (Silberberg & Lesker, 1975; Silberberg, Hasler & Lesker, 1976).
Stumpy is yet another genetically distinct chondrodystrophic dwarf with adistinctive cartilage phenotype.
1 Author's address: Department of Anatomy, School of Medicine, University of Leeds,Leeds LS2 9NL, U.K.
280 D. R. JOHNSON
Ultrastructural observations on stumpy (STM) 281
ORIGIN AND GENETICS
Stumpy arose in Cambridge in the control series of an experiment designed totest the mutagenicity of oral contraceptives. It is not allelic with achondroplasia(en), brachymorphic (bm), stubby (stb) (Ferguson & Wallace, 1973) nor cartilageanomaly (can) (D. R. Johnson, unpublished).
The gene behaves as a simple recessive: female stmjstm are good mothers,but males are usually sterile. An account of the genetics and linkage of stumpywill appear elsewhere.
MATERIAL AND METHODS
The left knee and elbow regions of fourteen stmjstm mice aged 5-14 days and12 normal litter-mates were fixed in Bouin's fluid, dehydrated, embedded inparaffin wax, sectioned longitudinally at 8 /on and stained with haematoxylinand eosin. The right knee and elbow were fixed in cacodylate-sucrose bufferedglutaraldehyde at pH 7-2, post-fixed in osmic acid, dehydrated and embeddedin Araldite or Spurr's resin. Ultrathin sections obtained with an LKB ultratomewere stained with uranyl acetate and lead citrate and examined in an AEIEM6B electron microscope.
RESULTS
Light microscopy of immature stmjstm long bones shows reduced stainingof the cartilage matrix. The number of cells per lacuna is increased, and aslight increase (14%) is seen in the number of mitotic figures. The zone ofhypertrophy is extensive: hypertrophied cells seem to be a little larger than innormal litter-mates, and are surrounded by a dense rim of matrix (Figs. 1 and2).
Electron microscopy shows normal-looking cells set in a normal-lookingmatrix (Figs. 3 and 4). However, a larger number of cells than normal lie inclose approximation to their neighbour, and nests of four or eight adjoiningcells were often seen. When two cells are adjacent, contact between them ismaintained either by large areas of membrane which remain in close proximitybut with a narrow intercellular gap, or, less commonly, by tight junctions(Figs. 5 and 6). In some cells, especially those from older (14-day) animals,the junctional region was complicated by folds in the membranes (Figs. 7
FIGURES 1-4
Figs. 1, 2. Sections through lower femoral epiphyseal plates of + /+ or +lstm andstmjstm mice respectively. In Fig. 2, note the wide zone of hypertrophy, densedeposition of matrix around hypertrophic cells and increased number of cellsperlacuna.Fig. 3. stmlstm chondrocytes: note normal appearance of cells and matrix.Fig. 4. stmlstm chondrocytes showing areas of membrane contact.
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FIGURES 5-8
Fig. 5. Tight junction between stmlstm chondrocytes.Fig. 6. Area of contact between stmlstm chondrocytes showing close apposition ofmembrane and vesicles (V).Figs. 7, 8. Tortuous membrane between stmlstm chondrocytes.
Ultrastructural observations on stumpy (STM) 283and 8), which varied from simple peg and socket arrangements to complex inter-digitations.
Although occasional chondrocytes (supposed to be the products of a recentdivision) were seen lying adjacent to one another in normal controls, tightjunctions and membrane folding were not seen.
DISCUSSION
The main abnormality noted in stm cartilage appears to concern the failureof chondrocytes to move apart after mitosis. In normal sibs of stm mice,chondrocytes were seen side by side only rarely, and, in these cases, no involve-ment of membranes was seen.
The presence of large numbers of adjacent cells suggests a burst of mitosisand the mitotic rate in stm cartilage is slightly elevated. Also, I found (Johnson,1977) that stumpy femora and tibiae are growing at normal rates or slightlyfaster over the period covered by the present observations: an abrupt cessationof growth follows at around 16 days.
Somewhat similar chondrocytes were described by Fahmy, Lee & Johnson(1971) in rat cartilage exposed to testosterone. These cells showed a high mitoticrate with many closely apposed cells, well developed secretory activity and areasof membrane contact between adjacent cells. Fahmy found that testosteroneserved to accelerate the processes of division, maturation, hypertrophy anddegeneration. This is in accordance with the well documented effects of steroidhormones; in general, small doses accelerate bone growth, large doses accelerategrowth to such an extent that premature closure of the epiphyses occurs.However, Fahmy et al. did not describe tight junctions nor intercalations ofmembrane, which appear to be unique to the stm phenotype. The increase inmitosis, coupled with the wide zone of hypertrophy (Fig. 2), suggests thepossibility of a temporary hormone imbalance in growing stm mice. In thiscase, the presence of male sterility may assume significance.
Stumpy fits well into the emerging picture of chondrodystrophy, a series ofshort-legged phenotypes produced by anomalies in cartilage metabolism. Thenature of these anomalies appears variable: at present, there seem to be almostas many of them as there are mutants described. Perhaps we should be askingourselves not what are the biochemical steps involved in the production ofabnormal cartilage (although this knowledge will surely prove to be of value inthe study of normal cartilage metabolism) but rather why cartilage is sovulnerable.
Consider as an example the achondroplastic (ac) rabbit. Bargman et al.(1972) showed that the primary defect in these animals is a failure to generateATP at the cytochrome oxidase region (site III) of the terminal respiratorychain during oxidative phosphorylation. This mitochondrial defect must bepresent in all cells of the homozygous abnormal animal, and was, in fact,
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demonstrated in liver mitochondria. Yet the most striking feature of the ac/acrabbit is its achondroplasia. Bargman et al. suggest that, in tissues such ascartilage where oxygen tension is normally low, oxidative energy formationmay be rate-limiting for growth.
It seems likely that this will not prove to be the only example of a rate-limiting process in cartilage: perhaps the present wide spectra of biochemicalbases for achondroplasia have this in common, that they all represent break-downs in rate-limiting steps on one of the many metabolic pathways present inthe relatively isolated chondrocyte.
REFERENCES
BARGMAN, G. J., MACKLER, B. & SHEPARD, T. H. (1972). Studies of oxidative energy de-ficiency. I. Achondroplasia in the rabbit. Archs Biochem. Biophys. 150, 137-146.
FAHMY, A., LEE, S. & JOHNSON, P. (1971). Ultrastructural effects of testosterone on epiphysealcartilage. Calcified Tiss. Res. 7, 12-22.
FERGUSON, J. M. & WALLACE, M. E. (1973). Personal communication. Mouse News Letter49, 23.
FRASER, R. A. & GOETINCK, P. F. (1971). Reduced synthesis of chondroitin sulphate bycartilage from the mutant nanomelia. Biochem. biophys. Res. Comm. 43, 494-503.
JOHNSON, D. R. (1977). The growth of femur and tibia in three genetically distinct chondro-dystrophic mutants in the mouse. / . Anat. (in press).
JOHNSON, D. R. & HUNT, D. M. (1974). Biochemical observations on the cartilage of achon-droplastic (can) mice. / . Embryol. exp. Morph. 31, 319-328.
ORKIN, R. W., PRATT, R. M. & GILL, M. (1976). Undersulphated chondroitin sulphate inthe cartilage matrix of brachymorphic mice. Devi Biol. 50, 82-94.
PENNYPACKER, J. P. & GOETINCK, P. J. (1976). Biochemical and ultrastructural studies ofcollagen and proteochondroitin.sulphate in normal and nanomelic cartilage. Devi Biol.50, 35-47.
RIMOIN, D. L. (1975) The condrodystrophies. Adv. hum. Genet. 5, 1-118.SEEGMILLER, FRASER, F. C. & SHELDON, H. (1971). A new chondrodystrophic mutant in
mice. / . Cell Biol. 48, 580-593.SEEGMILLER, R., FERGUSON, C. C. & SHELDON, H. (1972). Studies on cartilage. VI. A genetically
determined defect in tracheal cartilage. / . Ultrastruct. Res. 38, 288-301.SILBERBERG, R. & LESKER, P. (1975). Skeletal growth and development of achondroplastic
mice. Growth 39, 17-37.SILBERBERG, R., HASLER, M. & LESKER, P. (1976). Ultrastructure of articular cartilage of
achondroplastic mice. Acta anat. 96, 162-175.
(Received 15 November 1976, revised 17 January 1977)
