Bone metabolism in the osteopetrotic rat mutation microphthalmia blanc

Bone Vol. 16, No. 5 May 1995:567-574 ELSEVIER

Bone Metabolism in the Osteopetrotic Rat Mutation Microphthalmia Blanc M. J. CIELINSKI and S. C. M A R K S , JR. Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA

(Marks et al. 1984; Seifert et al. 1993). Because this heterogeneity is mirrored in humans with respect to clinical manifestations (Cournot et al. 1992; Monaghan et al. 1991) and treatment (Coccia 1993), the continued study of animal mutations may lead to valuable insights into human diseases and possible clinical treatments. A new spontaneous osteopetrotic mutation called microphthalmia blanc (mib) in the rat has been reported, in which affected individuals are born with a typical sclerotic skeleton that improves with age (Moutier et al. 1989). It is interesting that this disease progression is similar to that reported for an infant (Monaghan et al. 1991) who was born with radiographic evidence of severe osteopetrosis, which gradually diminished during the 1st year of life. Our recent studies have shown that osteoclast numbers and histochemical staining for its functional enzymes tartrate-resistant acid phosphatase (TRAP) and tartrateresistant acid ATPase (TrATPase) are reduced in newborn mib rats and that these parameters improve to near-normal levels by the 4th postnatal week. These data indicate that the mild, transient osteopetrosis in the mib rat is caused by neonatal reductions in osteoclast number and function (Cielinski & Marks 1994). In this study we examined bone metabolism in mib rats, including the rate of bone formation and the ultrastructural and molecular characteristics of osteoclasts, to explore further the mild, transient nature of osteopetrosis in this mutation.

We have examined parameters of bone metabolism in a new mutation, microphthalmia blanc (mib), in the rat exhibiting a skeletal sclerosis at birth that improves with age. There were no significant differences in the rate of bone formation during the first postnatal month except a temporary reduction in mutants at 3 weeks that coincided with compromised nutrition at weaning. At birth the ruffled border in mutant osteoclasts was absent or poorly developed and mRNA analyses of mutant bone compared to normal bone showed significant reductions in the messages for the osteoclast-specific genes carbonic andydrase II and tartrate-resistant ATPase. These distinctive uitrastructural and molecular differences were not present 1 month later. These data show that the transient osteopetrosis in mib rats results from a perinatal reduction in ultrastructural and enzymatic features of active osteoclasts and is not complicated by elevations in bone formarion. The molecular basis for both the production and resolution of these abnormalities deserves further study.

(Bone 16:567-574; 1995) Key Words: Osteopetrosis; Osteoclast; Microphthalmia blanc; Gene expression; Bone formation; Bone resorption; Ultrastructure.

Introduction Materials and Methods Osteopetrosis is a rare metabolic disease of bone caused by reduced bone resorption and distinguished by a generalized skeletal sclerosis, absent or reduced marrow cavities, and impaired tooth eruption (Marks 1987; Osier & Marks 1992). Among mammals the disease is congenital, occurs spontaneously in a variety of species (Zetterholm 1972; Lees & Sautter 1979; Smits & Bubenik, 1990; Gopal et al. 1980) including humans (LoriaCortes et al. 1977), and is inherited as an autosomal recessive trait. Studies of the nine separate mutations in common laboratory animals have shown that the disease state is manifested differently in each (Seifert et al. 1993). Although osteopetrosis is caused by abnormal osteoclast development and/or function (Marks 1987; Marks & McGuire 1988), differences in osteoclast number, calcium and phosphorous homeostasis, serum levels of 1,25-dihydroxyvitamin D3, degree of marrow cavity formation and tooth eruption, responsiveness to cure by stem cell replacement, and general prognosis exist among the different mutations

Animals Animals used in this study were the offspring of tested females (+/mib) and mutant males (mib/mib) derived from breeding stock donated by Dr. Ren6 Moutier (Center de S61ection et d'Elevage d'Animaux de Laboratorie CNRS, Orl6ans, France). The animals were housed, maintained, and used at the University of Massachusetts according to the NIH guide for the care and use of laboratory animals (1985) and the guidelines and recommendations of the University of Massachusetts Animal Advisory Committee. Homozygous microphthalmia blanc (mib/mib) rats were identified at birth by a lack of pigmentation. Ultrastructural Observations At birth and 7 weeks, a minimum of four normal and four mib/ mib littermates were decapitated and their tibiae and femora rapidly removed, cleaned of soft tissue, transected longitudinallyto facilitate penetration of fixative, and fixed in 4% gluteraldehyde in 0.1M sodium cacodylate buffer, pH 7.2, for 1.5 h at 4°C. Fixed specimens were rinsed in 0.1M sodium cacodylate buffer, de-

Address for correspondence and reprints: Dr. Matthew J. Cielinski, Department of Cytokine Biology, Forsyth Dental Center, 140 The Fenway, Boston, MA 02115. © 1995by ElsevierScienceInc.

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mineralized in 10% EDTA in 10 mM Tris buffer, postfixed in 1% osmium tetroxide, dehydrated in a graded ethanol series, and embedded in epon. Semithin sections (1 Ixm) were stained with toluidine blue to study cytologic details before cutting thin sections for transmission electron microscopy. This sections were collected on 200-mesh grids and stained with 8% uranyl acetate and 2% lead citrate. We examined 20 normal and 22 mib/mib osteoclasts by transmission electron microscopy. Northern Analyses

Total cellular RNA was isolated from long bones (tibiae and femora) by a modification of the Chirgwin procedure (Chirgwin et al. 1979) as previously described (Shalhoub et al. 1991). Briefly, long bones were removed from newborn and 4-week-old animals (decapitated), cleaned of excess soft tissue, snap-frozen in liquid nitrogen, and stored at - 70°C until use. At 4 weeks, a minimum of four normal and four mib/mib rats were used and the long bones of newborn rats were pooled according to genotype from a minimum of five litters (each consisting of a least three normal and three mib/mib rats). Bone samples were crushed into powder using a Bessman tissue pulverizer (Fisher, Springfield, NJ) precooled in liquid nitrogen. Total RNA was extracted (5M guanidine thiocyanate with 2% sarcosyl and 72 mM [3-mercaptoethanol) and recovered as a translucent pellet after centrifugation through a 5.7M cesium chloride gradient. This RNA pellet was resuspended (10 mM Tris, pH 7.4, and 5 mM EDTA), precipitated with 2.5 vol ethanol, partially dried under vacuum, and resuspended and precipitated a second time in TE buffer and 2.5 vol of ethanol. RNA was resuspended in ultrapure water (Sigma), quantitated by absorbence at 260 nm, and stored at - 7 0 ° C . The quality of RNA was monitored with respect to representation of 28S and 18S ribososomal RNA as an internal standard, and the intactness monitored by electrophoretic fractionation (1% agarose gels, 6.6% formaldehyde, ethidium bromide staining). RNA samples 10-15 I.Lg) were then transferred by capillary action to Genescreen Plus hybridization transfer membranes (Dupont, NEN Research Products, Boston, MA) and cross-linked to the membrane by ultraviolet (UV) light exposure for 1.5-2.0 min, and the membranes were stored at 4°C in sealed plastic bags. Probes were labeled with [o~-32p]dCTP by the random primer method (Feinberg and Vogelstein 1983) to a specific activity of at least 1 x 1 0 9 dprn/ixg DNA). Prehybridization and hybridization of membranes were performed in 50% formamide, 5 × SSC, 10x Denhardt's solution, 50 mM sodium phosphate (pH 6.5), 1% sodium dodecyl sulfate (SDS), and 50 I~g/ml ss-salmon sperm DNA (Sigma) for 24 h at 42°C. Blots were washed at 3 x 20 min with 2× SSC, 0.1% SDS at 50°C, 2 x 20 min with I x SSC, 0.1% SDS at 50°C, and once with 0. I x SSC, 0.1% SDS at 65°C. The blots were then exposed to X-OMAT AR X-ray film (Eastman Kodak, Rochester, NY) using a Cronex Lightning Plus intensifying screen at - 70°C. The resultant autoradiograms were quantitated by scanning laser densitometry (LKB 2400 GelScan XL) within a linear range of signals. All data obtained were normalized with respect to 18S ribosomal RNA and expressed in densitometric units; statistical significance was defined as differences with a probability <0.5% (two-tailed Student test).

Bone Vol. 16, No. 5 May 1995:567-574 body weight at birth and at weekly intervals during the 1st postnatal month (Walker 1966). Rats were killed 6 h later and the calvariae were removed, cleaned of excess soft tissue, air-dried, weighed, minced into a fine powder with scissors, and subjected to digestion with 1M HC1 at 60°C overnight. Samples were then neutralized with an equivalent volume of 1M NaOH, mixed with l0 ml scintillation fluid, and placed in a scintillation counter. These measurements of [3H]proline in bone samples have been shown to be reliable indices of [3]hydroxyproline and bone formation in normal and osteopetrotic animals (Marks 1969). Data were normalized to counts per minute per gram calvarial bone, and the rate of bone formation for mutants at each age was expressed as a percentage of the data from normal littermates. Results

Table 1 shows the rate of bone formation in rats of mib stock at 1, 7, 14, and 28 days determined by quantitative analysis of the incorporation of radiolabeled proline into newly formed bone matrix. Bone formation fell within normal ranges for all ages observed, with the exception of 21 days, when bone formation was reduced in mib/mib rats to 56-60% compared to normal littermates. The ultrastructural features of osteoclasts in tibiae of normal and mutants rats of mib stock analyzed at birth and 7 weeks are shown in representative transmission electron micrographs in Figures 1, 2, and 3. At birth all normal osteoclasts exhibited features characteristic of actively resorbing cells (Figure l a), including highly developed ruffled borders flanked by clear zones and abundant mitochondria, free ribosomes, and cytoplasmic vacuoles. The three representative mutant osteoclasts at birth shown in Figures la and 2 illustrate that although these cells possessed clear zones, numerous mitochondria, cytoplasmic vacuoles, and free ribosomes, the degree of membrane ruffling varied from nonexistant (Figure 2a) to minimal (Figures la and 2b) in mib/mib rats. Figure 2b shows the best ruffled border we observed in mutant osteoclasts at birth. At 7 weeks all normal osteoclasts displayed the features of resorbing cells (Figure 3a), and mutant osteoclasts possessed numerous vacuoles, mitochondria, and distinct clear zones surrounding distinct ruffled borders (Figure 3b). Figure 4 is a representative pair of autoradiograms of the same Northern blot of total cellular RNA from litters of newborn rats of mib stock hybridized with B2[p]dCTP-labeled probe for tartrate resistant acid ATPase (TrATPase) and 18S ribososomal RNA. These and similar Northern blots were used to generate data presented in densitimetric units and normalized to 18S ribososomal RNA, as summarized in Figure 5. The osteoclastspecific messages TrATPase and carbonic anhydrase II (CA-II) were significantly reduced in newborn mib/mib rats compared to normal littermates. The relative abundance of skeletal TrATPase Table 1. Bone metabolism in rats of mib stock a

Age (days)

Osteoclasts (%)b

Bone formation (%)c

48 55 46 60 70

85 (74-99) 104 (99-112) 111 (103-119) 58 (56-60) 100

Bone Formation

1 7 14 21 28

To quantitate bone formation at least five mutant and five normal littermates of mib stock were injected intraperitoneally with 5.0 ~LCi [3H]proline (New England Nuclear, Boston, MA) per gram

aData for mutants expressed as a percentage of those for normal rats. bData from Cielinski and Marks (1994). CAverage (and range) of at least three measurements.

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Figure 1. Transmission electron micrographs of representative osteoclasts from l-day-old normal (A) and mib/rnib (B) rats. Normal osteoclasts exhibit characteristics of active osteoclasts including mitochondria (m), vacuoles (v), and a well-developed ruffled border (RB) surrounded by an organelle-free clear zone (cz). Mutant osteoclasts (b) possess mitochondria (m), vacuoles (v) and clear zones (cz), but membrane ruffling is limited (arrows). N, Nucleus; n, nucleolus; b, bone; c, cartilage; x9200.

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Figure 2. Transmission electron micrograph of osteoclasts from 1-day-old rnib/mib rats showing varying degrees of membrane ruffling. Mutant osteoclasts lack ruffled borders (A) or at best exhibit limited membrane ruffling (B, arrow), v, Vacuoles; N, nucleus; b, bone; c, cartilage; cz, clear zone; × 7450.

4

B

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Figure 3. Transmission electron micrographs of the ultrastructural features of representative active osteoclasts from 7-week-old normal (A) and mib/ rnib (B) littermates. In normal rats the cytoplasm of osteoclasts contains numerous mitochondria (m) and vacuoles (v) near the ruffled border (R). In the mutant osteoclast (B), a distinct ruffled border (R) is present adjacent to mineralized cartilage (c). cz, Clear zone; b, bone; N, nucleus; ×4000.

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n

n

n

n

n TrATPase

18S

® Figure 4. Representative Northern blot of total cellular RNA from l-day-old mib/mib and normal (n) rat long bones isolated and processed as described in the text. Top: Autoradiogram of a Northern blot hybridized with 32[P]dCTP-labeled probe for tartrate-resistant acid ATPase (1 week exposure). Notice the difference in the relative abundance of TrATPase message in normal rats (n, lanes 5, 6, 10-12) and mib/mib littermates (unlabeled, lanes 1-4, 7-9). Bottom: Autoradiogram of the same Northern blot probed for 18S ribososomal RNA as an internal standard.

and CA-II mRNA in normal and mib/mib rats did not differ at 4 weeks.

Discussion Previous studies have shown that rnib is a relatively mild mutation in which the skeletal sclerosis present at birth gradually diminishes by the 4th postnatal week as a result of neonatal reductions in osteoclast numbers and function (Moutier et al. 1989; Cielinski and Marks 1994). In this study we have examined bone metabolism including rate of bone formation, osteoclast numbers, and the ultrastructural and molecular characteris-

Northern Analysis of Osteociast-Specific Gene Expression in Rat Long Bones 3.0-

[] NLM

[] mib/mib

b.

2.0

E~ o'E e-

0.0

ATPase 1

ATPase 28

CA

mRNA and Age (days)

1

CA II 28

(~)

Figure 5. Results from Northern analyses of osteoclast-specific gene expression in rat long bones. Data are presented in densitometric units and normalized to 18S ribosomal RNA generated by scanning laser densitometry of blots from at least five litters (1 day) or four rats (28 days) of each genotype. Notice that at birth normal rats have greater amounts of both TrATPase and CA-If mRNAs. No differences between normal and mib/mib rats were observed at 28 days.

tics of osteoclasts in the mib rat. Osteoclasts of normal rats at birth and 7 weeks had the classical features of active resorbing cells including extensive ruffled borders, clear zones, and abundant mitochondria and free ribosomes (Holtrop & King 1977; Holtrop 1991). However, ruffled borders were absent or poorly developed in osteoclasts from newborn mib/rnib rats. By 7 weeks, mutant osteoclasts exhibited the usual features of actively resorbing cells with distinct membrane rufflings flanked by clear zones. These results indicate that the neonatal abnormalities in osteoclast function in mib/mib rats include the inability to form a well-developed ruffled border, similar to the situation in oc and mi mice, the os rabbit, and ia rat mutations (Marks 1984; Seifert et al. 1993). This defect was also resolved after the 1st postnatal month, similar to derrangernents in osteoclast numbers and function described earlier (Cielinski & Marks 1994). Osteoclast gene expression, as assayed by Northern analysis of total cellular RNA isolated from tibiae and femora, is also abnormal in neonatal mib/mib rats, as evidenced by significant reductions in mRNA encoding two important functional enzymes TrATPase and CA-II. These abnormalities also normalize by 4 weeks. These data agree with our previous study in which the abnormally low amounts to TrATPase and the related functional enzyme tartrate-resistant acid phosphatase (TRAP) detected by histochemical staining in neonatal mib/mib rats normalized by 4 weeks (Cielinski & Marks 1994). In addition, we examined the rate of bone formation in mib rats at 1, 7, 14, 21, and 28 days by quantitive analysis of the incorporation of radiolabeled proline into newly formed bone matrix (Walker 1966; Marks 1969). Bone formation fell within normal ranges for all ages except 21 days, when bone formation was reduced in mib/mib rats to 56--60% compared to normal littermates. We interpret this result to be a consequence of the nutritional disadvantage of mib/mib rats at weaning (3 weeks), when they must switch to a diet of ground food with rnaloccluded incisors and delayed eruption of molars (Cielinski et al. 1994). These data show that the mild osteopetrosis in mib rats is not complicated by increased bone formation as in some other mutations (Marks & McGuire 1988). Therefore, we conclude that the mild nature of osteopetrosis in mib rats is caused by reductions in both numbers and function of osteoclasts, which resolve during the 1st postnatal month. These abnormalities include low osteoclast numbers, reduced message encoding the functional enzymes TrATPase and CA-II, reduced production of the functional enzymes TRAP and TrATPase as shown by histochemical staining, and inability of osteoclasts to form ruffled borders. All these abnormalities are improved dramatically by 4 weeks or

Bone Vol. 16, No. 5 May 1995:567-574 later. These data are understandable given the transient nature of osteopetrosis in mib rats and the essential contributions of ruffled border formation and both TrATPase and CA-II in creating the microenvironment necessary for bone resorption (Baron et al. 1985; Gay 1992). Indeed, expression of carbonic anhydrase mRNA has recently been linked to resorbing osteoclasts (Asotra et al. 1994). The heterogeneity that exists among the mammalian osteopetroses, including humans, has been well documented (Marks et al. 1984; Cournot et al. 1992). Although the ia rat and the op mouse are two other osteopetrotic animals with severe neonatal osteopetrosis that improve later in life (Marks 1976, 1982, 1987), they differ with respect to osteoclast numbers, histochemical staining, extent of ruffled borders, and rates of bone formation. In the ia rat, afflicted individuals possess increased numbers of osteoclasts that stain heavily for the functional enzyme TRAP but fail to elaborate a ruffled border (Marks 1976). The op mouse has reduced numbers of osteoclasts, but those present have highly developed ruffled borders (Marks 1982). Taken together with our data concerning the mib rat, it is clear that even among the milder forms of osteopetrosis there exists considerable heterogeneity. We have only begun to elucidate the various molecular mechanisms responsible for the common disease state known as osteopetrosis. Recent investigations illustrate the importance of cytokines and components of the intracellular signaling pathways in normal osteoclast development and function. Studies concerning the op mouse and tl rat have illustrated the importance of the hemopoietic growth factor colony-stimulating factor-1 (CSF-1) in normal osteoclast development (Felix et al. 1990; Kodama et al. 1991; Marks et al. 1992). However, it is noteworthy that CSF- 1 treatment of op mice and tl rats does not completely cure afflicted animals of osteopetrosis, as residual sclerosis and failure to restore osteoclasts in the subepiphyseal regions of the long bones persist (Marks et al. 1993; Sundquist et al. 1995). In addition, genetic disruptions or "knockouts" of the tyrosine kinase c-src and the c-fos component of the AP-1 transcription factor complex have resulted in osteopetrosis in mice (Soriano et al. 1991; Grigoriadis et al. 1994). These observations--the heterogeneity among the osteopetroses, and the emerging concept of redundancy in the genome (Erickson 1993)---suggest that aberrant expression of significant components involved in important signal pathways could give rise to a common disease state and account for the variability of clinical symptoms observed. Accordingly, it is likely that studies using animals with osteopetrosis will shed light on the cellular mechanisms of bone resorption (Marks 1984, 1989, 1992; Marks & Walker 1976) and that detailed examination of signaling mechanisms of bone cells from these animal models of osteopetrosis will elucidate the causes of the disease in animals and humans. Such studies may allow clinical investigators to differentiate between those infants born with osteopetrosis that need immediate treatment and those who do not. For example, we postulate that abnormalities in the Ras superfamily of signaling molecules (Pawson 1993; Boguski & McCormick 1993), in which Rac phosphorylation leads to actin membrane ruffling and Rho phosphorylation stimulates actin stress fiber formation, could directly account for the abnormalities in ruffled border formation observed in the mib rat and other mutations and indirectly account for decreased levels of the membrane-associated enzyme TrATPase. However, our current inability to isolate pure populations of mammalian osteoclasts, the cells in question, will make these studies difficult.

Acknowledgments: This work was supported by NIH Grant DE07444.

The authors thank Carole McKay, Erik Larson, and Cindy Richards for

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exceptional technical assistance, and April Mason-Savas for cutting the ultrathin sections for transmission electron microscopy. References Asotra, A., Gupta, A. K., Sodek, J., Aubin, J. E., and I-Ieersche, J. N. M. Carbonic anhydrase II mRNA expression in individual osteoclasts under "resorbing" and "nonresorbing'" conditions. J Bone Min Res 9:1115-1122; 1994. Baron, R., Neff, L., Louvard, D., and Courtoy, P. J. Cell mediated extracellular acidification and bone resorption: Evidence for a low pH in resorbing lacunae and localization of a 100-kD lycosomal membrane protein of the osteoclast ruffled border. J Cell Biol 101:2210-2222; 1985. Boguski, M. S. and McCormick, F. Proteins regulating Ras and its relatives. Nature 366:643-654; 1993. Chirgwin, J. H., Pryzybyla, A. E., MacDonald, R. J., and Rutter, W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299; 1979. Cielinski, M. and Marks, S. C., Jr. Neonatal reductions in osteoclast number and function account for the transient nature of osteopetrosis in the rat mutation microphthalmia blanc (mib). Bone 15:707-715; 1994. Cielinski, M., lizuka, T., and Marks, S. C., Jr. Dental abnormalities in the osteopetrotic rat mutation microphthalmia blanc. Arch Oral Biol 39:985-990; 1994. Coccia, P. F. Bone marrow transplantation for osteopetrosis. Forman, S. J., Blume, K. G., and Thomas, E. D., eds. Bone marrow transplantation. Boston, MA: Blackwell Scientific Publications; 1993;Chapter 65,874-882. Cournot, G., Trubert-thil, C. L., Petrovic, M., Boyle, A., Cormier, C., Girault, D., Fischer, A., and Garabedian. M. Mineral metabolism in infants with malignant osteopetrosis: Heterogeneity in plasma 1,25-dihydroxyvitamin D levels and bone histology. J Bone Min Res 7:1-10; 1992. Erickson, H. P. Gene knockouts of c-src, transforming growth factor 131, and tenascin suggest superfluous, nonfunctional expression of proteins. J Cell Biol 120:1079-1081; 1993. Feinberg, A. P. and Vogelstein, B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6-13; 1983. Felix, R., Cecchini, M. G., Hofstetter, W., Elford, P. R., Stutzer, A., and Fteisch, H. Macrophage colony stimulating factor restores in vivo bone resorption in the op/op osteopetrotic mouse. Endocrinology 127:2592-2594; 1990. Gay, C. V. Osteoclast ultrastructure and enzyme histochemistry: functional implecations, Rifkin, B. R., and Gay, C. V. eds. Biology and physiology of the osteoclast. Boca Raton, FL: CRC Press; 1992;130-150. Gopal, T., Leipold, H. W., and Dennis, S. M. Hydatidiform moles in Holstein cattle. Vet Rec 107:395-397; 1980. Grigoriadis, A. E., Wang, Z.-Q., Cecchini, M. G., Hofstetter, W., Felix, R., Fleisch, H. A., and Wagner, E. F. c-Fos: A key regulator of osteoclastmacrophage lineage determination and bone remodelling. Science 266:44344-8; 1994. Holtrop, M. E. Light and electron microscopic structure of osteoclasts. Hall, B. K., ed. Bone, vol. 2: The osteoclast. Boca Raton, FL: CRC Press; 1991;130. Holtrop, M. E. and King, G. J. The ultrastructure of the osteoclast and its functional implications. Clin Orthop 123:177-196; 1977. Kodama, H., Yamasaki, A., Nose, M., Nida, S., Ohgame, ¥ . , Abe, M., Kumegawa, M., and Suda, T. Congenital osteoclast deficiency in osteopetrotic (op/op) mice is cured by injections of macrophage colony-stimulating factor. J Exp Med 173:269-272; 1991. Lees, G. E. and Sautter, J. H. Anemia and osteopetrosis in a dog. J Amer Vet Med Assoc 175:820-824; 1979. Loria-Cortes, R., Quesada-Calvo, E., and Cordero-Chaverri, C. Osteopetrosis in children: A report of 26 cases. J Pediatr 91:4347; 1977. Marks, S. C., Jr. The specificity of the 3H-proline incorporation test as a measure of bone matrix formation. Biochem Biophys Res Commun 35: 236-242; 1969. Marks, S. C., Jr. Pathogenesis of osteopetrosis in the ia rat: Reduced bone resorption due to reduced osteoclast function. Am J Anat 138:165-190; 1976. Marks, S. C., Jr. Morphological evidence of reduced bone resorption in osteopetrotic (op) mice. Am J Anat 163:157-167; 1982. Marks~ S. C., Jr. Congenital osteopetrotic mutations as probes of the origin, structure, and function of osteoclasts. Clin Orthop 189:239-263; 1984. Marks, S. C., Jr. Osteopetrosis--multiple pathways for the interception of osteoclast function. Appl Pathol 5:172-183; 1987.

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M . J . Cielinski a n d S. C. M a r k s , Jr. B o n e m e t a b o l i s m in mib rats

Marks, S. C., Jr. Osteoclast biology: lessons from mammalian mutations. Amer J Med Gen 34:43-54; 1989. Marks, S. C., Jr. Congenital osteopetrosis in mammals--new windows on osteoclast biology. Slavkin, H., and Price, P., eds. Chemistry and biology of mineralized tissues. Amsterdam: Elsevier Science Publishers; 1992; 475-482. Marks, S. C., Jr., and McGuire, J. L. Primary bone cell dysfunction I1-osteopetrosis. Tam, C. S., Heersche, J. N. M., and Murray, T. M., eds. Metabolic bone disease: Cellular and tissue mechanisms. Boca Raton, FL: CRC Press; 1988;4%61. Marks, S. C., Jr. and Walker, D. G. Mammalian osteopetrosis: A model for studying cellular and humoral factors in bone resorption. Bourne, G., ed. The biochemistry and physiology of bone, vol 3. New York: Academic Press; 1976; 227-301. Marks, C. R., Seifert, M. F., and Marks, S. C., III. Osteoclast populations in congenital osteopetrosis: Additional evidence of hetergeneity. Metabol Bone Dis 5:259-264; 1984. Marks, S. C., Jr., Wojtowicz, A., Szperl, M., Urbanowska, E., MacKay, C. A., Wiktor-Jedrzejczak, W., Stanley, E. R., and Aukerman, S. L. Administration of colony-stimulating factor-1 corrects some macrophage, dental and skeletal defects in an osteopetrotic mutation (toothless, tl) in the rat. Bone 13:89-93; 1992. Marks, S. C., Jr., MacKay, C. A., Jackson, M. E., Larson, E. K., Cielinski, M. J., Stanley, E. R., and Aukerman, S. L. The skeletal effects of colony stimulating factor-I in toothless (osteopetrotic) rats; Persistent metaphyseal sclerosis and the failure to restore subepiphyseal osteoclasts. Bone 14:675-680; 1993. Monaghan, B. A., Kaplan, F. S., August, C. S., Fallon, M. D., and Flannery, D. B. Transient infantile osteopetrosis. J Pediat 118:252-255; 1991. Moutier, R., Ostrowski, K., and Lamendin, H. Microphthalmia: A new recessive mutation in the Norway rat. J Hered 80:76-78; 1989.

Bone Vol. 16, No. 5 May 1995:567-574 Osier, L. K. and Marks, S. C., Jr. Osteopetrosis. Rifkin, B. R., and Gay, G. V., eds. Biology and physiology of the osteoclast. Boca Raton, FL: CRC Press; 1992; 433~.54. Pawson, T. Signal transduction--a conserved pathway from membrane to nucleus. Devel Genet 14:333-338; 1993. Seifert, M. F., Popoff, S. N., Jackson, M. E., Mackay, C. A., Cielinski, M., and Marks, S. C., Jr. Experimental studies of osteopetrosis in laboratory animals. Clin Orthop Rel Res 294:23-33; 1993. Shalhoub, V., Jackson, M. E., Lian, J. B., Stein, G. S., and Marks, S. C., Jr. Gene expression during skeletal development in three osteopetrotic rat mutations: Evidence for osteoblast abnormalities. J Biol Chem 266:9847-9856; 1991. Smits, B. and Bubenik, G. A. Congenital osteopetrosis in white-tailed deer (Odocoileus virginianus). J Wildlife Dis 26:567-571; 1990. Soriano, P., Montgomery, C., Geske, R., and Bradley, A. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell 64:693 702; 1991. Sundquist, K. T., Cecchini, M. G., and Marks, S. C., Jr. CSF-1 injections improve but do not cure skeletal sclerosis in osteopetrotic (op) mice. Bone 16:3%46; 1995. Walker, D. G. Counteraction to parathyroid therapy in osteopetrotic mice as revealed in the plasmia calcium level and ability to incorporate 3H-proline into bone. Endocrinology 79:836-842; 1966. Zetterholm, R. Osteopetrosis and hyperstosis in cattle. Acta Radiologica (Suppl 319):107-116; 1972.

Date Received: December 9, 1994 Date Accepted: January 25, 1995