Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8214-8219, August 1996 Biochemistry

Targeted disruption of the B gene results in mice resembling the phenotype of VI (animal model/homologous recombination/lysosomal storage disease/Maroteaux-Lamy syndrome) MEIKE EVERS*, PAUL SAFTIG*, PETER SCHMIDTt, ANGELA HAFNERt, DANIELA B. MCLOGHLINt, WOLFGANG SCHMAHLt, BARBARA HESS*, KURT VON FIGURA*, AND CHRISTOPH PETERS*: *Zentrum Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Universitat Gottingen, 37073 Gottingen, Germany; and tInstitut fur Tierpathologie, Universitat Munchen, 80539 Munich, Germany Communicated by Elizabeth F. Neufeld, University of California School of Medicine, Los Angeles, CA, April 26, 1996 (received for review January 23, 1996)

ABSTRACT Mucopolysaccharidosis VI (MPS VI) is a documenting heterogeneity of the disease at the molecular lysosomal storage disease with autosomal recessive inheri- level (7). tance caused by a deficiency of the arylsulfatase B There is no effective therapy for MPS VI or any other MPS. (ASB), which is involved in degradation of Bone marrow transplantations have been performed in MPS and chondroitin 4-sulfate. A MPS VI mouse model was VI and other mucopolysaccharidoses with varying outcome generated by targeted disruption of the ASB gene. Homozy- (1). Since HLA matched donors are not available for the gous mutant animals exhibit ASB enzyme deficiency and majority of MPS patients, bone marrow transplantation is still elevated urinary secretion of dermatan sulfate. They develop associated with high mortality rates (1). Enzyme replacement progressive symptoms resembling those ofMPS VI in humans. (8) and somatic gene therapy (9) provide promising aspects for Around 4 weeks of age facial dysmorphia becomes overt, long therapy of lysosomal storage diseases, but suitable animal bones are shortened, and pelvic and costal abnormalities are models are required for their evaluation. Although cat (10) and observed. Major alterations in bone formation with perturbed rat (11) strains with ASB deficiencies have been described, cartilaginous tissues in newborns and widened, perturbed, they are not as appropriate for testing a variety of therapeutic and persisting growth plates in adult animals are seen. All strategies as a murine model. Here we report the generation of major parenchymal organs show storage of glycosaminogly- a MPS VI mouse model by disrupting the ASB gene (Asl-s) by cans preferentially in interstitial cells and macrophages. homologous recombination (12). Affected mice are fertile and mortality is not elevated up to 15 months of age. This mouse model will be a valuable tool for MATERIALS AND METHODS of MPS VI and to evaluate studying pathogenesis may help Isolation of a Genomic Clone and Targeting Vector Con- therapeutical approaches for lysosomal storage diseases. struction. A 758-bp partial murine ASB cDNA clone (pCABM750) was isolated from a Agtll brain cDNA library Mucopolysaccharidosis type VI (MPS VI) or Maroteaux- (Clontech) by homology screening with a full-length human Lamy syndrome is a lysosomal storage disease with autosomal ASB cDNA probe (4). The sequence of recessive inheritance. MPS VI has been described as a Morbus pCABM750 was deposited in the GenBank/EMBL data base Hurler (MPS I)-like syndrome, distinguished by normal men- (accession no. X92096). pCABM750 exhibits 81.8% sequence tal development. Patients with the severe form of MPS VI identity with the human ASB cDNA (4) on nucleotide and show growth retardation, dysostosis multiplex with macro- 83.1% on deduced amino acid level (data not shown). In cephaly, deformities of chest and vertebral bodies, pelvic EcoRI digested genomic DNA of 129/SvJ mice a 9-kbp malformation, and dysplasia of long bones. Restriction ofjoint and a 1.6-kbp DNA fragment are hybridizing with pCABM750. movement develops and claw-hand deformities are seen. The A subgenomic DNA library was constructed by ligation of 8- facies can be mildly affected or reach the coarseness charac- to 10-kbp EcoRI DNA fragments from genomic liver DNA of teristic of . Corneal clouding, hernias, a 129/SvJ with X-EMBL4 arms and subsequent packaging ac- thickened skin, and hepatosplenomegaly are observed. Car- cording to the instructions of the supplier (Stratagene). A diac involvement with valvular dysfunctions are common. MPS 9-kbp genomic clone (pmAB9E, see Fig. la) was isolated from VI patients with an intermediate or mild phenotype can be the library by hybridization with pCABM750. For construction differentiated from the severe course. Patients with severe of a targeting vector the 6-kbp BamHI DNA fragment from MPS VI often die of heart failure in their second or third pmAB9E was subcloned into pBlueskript SK-. A linker frag- decade. Spinal cord compression is a symptom frequently ment from the BamHI site to the Sall site of the plasmid vector observed in patients with milder forms of MPS VI (1). pUC18 was ligated to the XhoI site at the 5' end of the MPS VI is caused by a deficiency of the lysosomal enzyme neomycin (neo) expression cassette of pMClneo poly(A) (ref. arylsulfatase B (ASB; N-acetylgalactosamine-4-, EC 13; Stratagene); the neo cassette in pMClneo poly(A) contains 3.1.6.12). ASB hydrolyzes the sulfate ester group of N- a BamHI site at its 3' end. The neo cassette was inserted into acetylgalactosamine 4-sulfate residues of dermatan sulfate and the BglII site in exon 5 of the 6-kbp BamHI subclone described chondroitin 4-sulfate (2, 3). A deficiency of the enzyme causes above as a BamHI-BamHI restriction fragment resulting in the intralysosomal accumulation and urinary excretion of derma- targeting vector pAB6Bneo (see Fig. la). The insertion of the tan sulfate. The cDNA (4, 5) and the gene (6) of human ASB neo cassette introduces a premature translational stop codon have been isolated and characterized. Multiple mutations in the ASB genes of MPS VI patients have been detected, Abbreviations: ASB, arylsulfatase B; Asl-s, murine ASB gene; ES, embryonic stem; MPS, mucopolysaccharidosis; neo, neomycin. 4To whom reprint requests should be addressed at: Zentrum Bioche- The publication costs of this article were defrayed in part by page charge mie und Molekulare Zellbiologie, Abteilung Biochemie II, Univer- payment. This article must therefore be hereby marked "advertisement" in sitat Gottingen, Gosslerstrasse 12D, 37073 Gottingen, Germany. accordance with 18 U.S.C. §1734 solely to indicate this fact. e-mail: [email protected].

8214 Downloaded by guest on September 28, 2021 Biochemistry: Evers et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8215 into the ORF of the Asl-s gene after a deduced irrelevant tural gene (Asl-s) region was isolated by screening a sub- peptide of 82 amino acids. genomic 129/SvJ mouse library with a murine ASB cDNA Selection of Targeted Embryonic Stem (ES) Cells and probe (Fig. la; for details see Materials and Methods). The Generation of Mutant Mice. The targeting vector pAB6Bneo isolated segment of theAsl-s gene contains an ORF exhibiting was linearized with XhoI and introduced into the ES cell line 82% sequence identity on the nucleotide level with exon 5 of E14-1 (14) by electroporation. ES cells were cultured as the human ASB gene (data not shown). The targeting vector described, omitting gancyclovir selection (15). G418-resistant pAB6Bneo (Fig. la) was used for disruption of the ASB colonies were screened by Southern blot analysis of DNA structural gene (Asl-s) in ES cells. The ORF of the putative digested with EcoRI and hybridized with probe A (see Fig. la). exon 5 ofAsl-s is disrupted by insertion of a neo cassette. The ES cell clones with homologous recombination were con- ASB allele disrupted in exon 5 encodes a truncated polypep- firmed by digesting DNA with HindlIl or EcoRV and hybrid- tide lacking the C-terminal third of the ASB polypeptide. Since ization with probe A or probe B, respectively (see Fig. la). smaller C-terminal deletions of ASB have been shown to cause Mutated ES lines EB49 and EB68 were microinjected into the infantile form of MPS VI in humans (7), it may be blastocysts of C57BL/6J mice as described (15). Chimeric anticipated that the introduced mutation is a null allele. males were mated to C57BL/6J females. Mice were genotyped pAB6Bneo was introduced into E-14-1 ES cells (14) and for the Asl-s gene mutation by Southern blot analysis of G418-resistant colonies were analyzed by Southern blotting. BglII-digested genomic DNA, using probe A (see Fig. 1 a and Using the 3' external probe A (Fig. la) in 2 of 111 independent c). Homozygous mutant mice were obtained by mating het- clones tested an additional EcoRI DNA restriction fragment erozygous or homozygous mutant mice. was detected, indicating a homologous recombination in one Northern Blot Analysis. mRNA of from 4- to of the Asl-s gene alleles (Fig. lb). These results were con- 5-month-old mice was prepared from total RNA with Oligotex firmed by hybridizing HindIII-digested DNA with the same beads according to the instructions of the supplier (Qiagen, Chatsworth, CA). mRNA (3 jig) was separated in a formal- a dehyde agarose gel and processed as described (16). Filters E B H Bg HBg B BH E were hybridized with the murine ASB cDNA probe I I ,, l II Oxon 5 pCABM750 and glyceraldehyde-3-phosphate dehydrogenase probe 9 probe A (17), subsequently. B H Bg H E B ASB Assay and Determination of Urinary Glycosaminogly- 1I cans. Kidney homogenates from 8-month-old mice were gen- E--_ B H erated and processed as described (18). Aliquots representing E B H Bg H E B BH E 8 ,ug of protein were incubated with the trisaccharide substrate III I I N-acetylgalactosamine 4-sulfate-(1-4)-glucuronic acid-(1-3)- ne o & N-acetyl[3H]galactosaminitol-4-sulfate in the presence of 2-ac- etamido 2-deoxy-D-gluconolactone for 37 h at 37°C. Product 1 kb and substrate were separated by high voltage electrophoresis, and radioactivity was quantified by liquid scintillation counting as described (18). For determination of urinary glycosamino- glycans, 10 ,lI of urine with or without incubation with chondroitinase ABC or chondroitinase AC (Sigma) were b c dried, pellets were washed with methanol, resuspended in 0.5 Hindill EcoRI BgIll M acetic acid and spotted on DEAE membranes. Membranes 05 co were stained in 0.1% alcian blue in 0.5 M acetic acid for 15 min "gr (0 m CD m CD and destained in acetic acid. Absorbance at 480 nm was di UJ w w w w + + > measured (19). Pathology. For light microscopy, animals were sacrificed, organs were removed and fixed in 6% formaldehyde in phos- 9.0- phate-buffered saline (pH 7.4), and embedded in paraffin. 5.8- Sections (3 ,um) were prepared and stained with hematoxylin and eosin (20). For demonstration of , Mowry's colloidal iron or Alcian blue stain (performed at pH 1.4) were used (21). After removal of organs carcasses were pinned onto a cork sheet, fixed in formaldehyde as described, and radiographs were subsequently taken. For the analysis of epiphyseal and articular cartilage, the upper thigh and shin- bone were fixed in formaldehyde as described and embedded FIG. 1. Targeted disruption of the ASB (Asl-s) gene. (a) Strategy in Sections for inactivation of theAsl-s gene by homologous recombination in ES glycol methacrylate/methyl methacrylate. (2 ,um) cells. (I) Structure of genomic clone pmAB9E representing 9 kbp of were prepared and stained with hematoxylin and eosin. the Asl-s gene region. Exon 5 is indicated by a black box and flanking For electron microscopy mice were sacrificed, organs were introns are indicated by solid lines. Bars designated probe A and B removed, and tissue blocks (<1 mm3) were fixed in 6.25% denote DNA probes used for Southern blot analysis. (II) Targeting glutaraldehyde in S0rensen buffer, 67 mM, pH 7.4 at room vector pAB6Bneo with 6-kbp homology to the Asl-s gene locus. The temperature for 2 h. Samples were washed in S0rensen buffer neo cassette (open box) was inserted into a BglII restriction site in exon and postfixed in 1% osmium tetroxide in the same buffer at 4°C 5. Arrow marks direction of transcription of the neo gene, and the for 2 h. After dehydration in acetone samples were embedded broken line is the plasmid vector pBlueskript SK-. (III) Predicted in Epon. Thin sections were prepared and stained with uranyl Asl-s gene locus after homologous recombination. Restriction sites are acetate and lead citrate. as follows: E, EcoRI; B, BamHI; H, HindIII; Bg, BglII. (b) Southem blot analysis of ES cell clones. Probe A was hybridized to Hindlll- or EcoRI-digested genomic DNA from wild-type E-14-1 ES cells and RESULTS targeted ES cell clones (EB49 and EB68). For both an addi- tional 5.8-kbp DNA fragment indicates a targeted allele. (c) Southem Targeted Disruption of the ASB Gene and Generation of blot analysis of tail DNA. DNA was digested with BglII and analyzed Deficient Mice. A 9-kbp genomic clone from the ASB struc- with probe A (wild-type allele, 8.8 kbp; mutant allele, 11.5 kbp). Downloaded by guest on September 28, 2021 8216 Biochemistry: Evers et al. Proc. Natl. Acad. Sci. USA 93 (1996) probe A (Fig. lb) and by probing EcoRV digested DNA with the 5' external probe B (data not shown). Targeted ES cell a clones EB49 and EB68 were microinjected into blastocysts (12) and 15 chimeric mice (12 males and 3 females) were generated. Five of the chimeric males, derived from both ES cell lines, transmitted the mutated allele through the germline. Het- erozygous offspring was identified by hybridization of BglII digested DNA with probe A (data not shown). Heterozygotes exhibit a normal phenotype and normal fertility (data not shown). Genotyping of 290 offspring from heterozygote crosses (Fig. lc) originating from both microinjected ES cell clones revealed a frequency of 21.4% for homozygous mutant mice (Asl-s -/-), resembling the expected Mendelian frequency (25%). Hence, disruption of the Asl-s gene does not result in embry- onic lethality. Functional Inactivation of the ASB Gene. To test for ex- pression of the Asl-s gene in Asl-s - /- mice, Northern blot analyses were performed. A 4.2- and 1.9-kbp Asl-s-specific mRNA were detectable in kidney and liver mRNA from wild-type animals, whereas no Asl-s-specific transcripts were detectable in homozygous mutant animals (Fig. 2a). ASB enzyme activity can be differentiated from activities of other with the trisaccharide substrate N-acetylgalac- tosamine-4-sulfate-(1-4)-glucuronic acid-(1-3)-N-acetyl[3H]- galactosaminitol-4-sulfate (18). ASB activity was not detect- able in kidney, liver, spleen, and brain homogenates fromAsl-s -/- animals, whereas it was readily detectable in the respec- tive homogenates from wild-type mice (Fig. 2b; data shown for kidney only). Northern blot analysis and determination of ASB activity show that the Asl-s gene has been inactivated and that homozygous mutant mice are devoid of ASB enzyme activity. Phenotype of ASB-Deficient Mice. At birth and during the first postnatal weeks no overt phenotypic alterations were a b

E x

- Z.-E 0 coa) 0 (0 (- FIG. 3. Phenotype of ASB-deficient mice resembles human MPS co VI. (a) A 15-month-old Asl-s -/- mouse (Right) exhibits facial dismorphia with short nose and broad, flattened face and short fore limb. A wild-type littermate (Left) is shown for comparison. (b) Radiograph of carcasses of a 4-month-old Asl-s - / - mouse (- /-) + and a wild-type littermate (+/+). Multiple abnormalities including + + skull und pelvic abnormalities, shortened and thickened long bones and ribs and persisting growth plates most prominent in tail vertebrae FIG. 2. The ASB gene is inactivated in homozygous mutant mice. are demonstrable in the affected mouse and resemble features of (a) Northern blot analysis ofAsl-s mRNA expression. Kidney and liver dysostosis multiplex in human MPS VI. mRNA were hybridized to the partial murine ASB cDNA probe pCABM750 and murine glyceraldehyde-3-phosphate dehydrogenase detected inAsl-s - /- mice. Around 4 weeks of ageAsl-s -/- (G3PD; internal control). Asl-s-specific mRNA was undetectable in mice were distinguishable upon visual inspection from wild- mRNA from kidney and liver ofAsl-s - / - mice (-/-). The positions of 28 S and 18 S rRNA are indicated. (b) ASB enzyme activity. Kidney type controls. Facial dysmorphia characteristic for mucopo- homogenates from 8-month-old homozygous mutant mice (-/-) and lysaccharidoses with a broadened head and a shortened nose wild-type controls (+/+) were incubated with the specific trisaccha- became obvious. Limbs were shorter and paws were coarse and ride substrate N-acetylgalactosamine 4-sulfate-(1-4)-glucuronic acid- broad (Fig. 3a). Symptoms progress and the body weight of a in presence of 2-ac- (1-3)-N-acetyl[3H]galactosaminitol-4-sulfate the group of 9- to 12-month old Asl-s -/- mice was about 15% etamido 2-deoxy D-gluconolactone. The amount of product generated lower both for male and female animals in comparison to an was determined as described, and ASB enzyme activity was calculated. In kidney homogenates from homozygous mutant mice the ASB- age-matched wild-type control group (data not shown). In specific activity is at the limit of detection (n = 3 for -/- mice; n = affected animals urinary secretion of glycosaminoglycans was 4 for +/+ mice; SEM is given). elevated to 26.2 ± 5.4 ,tg/ml (age-matched controls, 3.7 ± 1.2 Downloaded by guest on September 28, 2021 Biochemistry: Evers et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8217 ,tg/ml). The majority of the urinary glycosaminoglycans from MPS VI mice were resistant to chondroitinase AC but not to chondroitinase ABC digestion and hence identified as derma- tan sulfate (data not shown). In blood smears coarse granular inclusion bodies equivalent to the Alder-Reilly bodies in human MPS VI (21) were observed in almost all polymorpho- nuclear leukocytes and in about 50% of the lymphocytes (data not shown). Corneal clouding was not observed. Asl-s -/- mice showed normal fertility, and no increase of mortality was observed up to an age of 15 months. Radiographs show that facial dysmorphia inAsl-s -/- mice is at least in part due to abnormalities of skull bones, since maxillae are shortened and the biparietal diameter of the skull is enlarged (Fig. 3b). In affected animals long bones are shortened and appear broadened, which is most obvious for humeri and femora (Fig. 3b) and has independently been confirmed by length determination of humeri and femora + upon autopsies of mice sacrificed at 3-12 months of age (Table +1 I' 1). Ribs exhibit thickening and wavy, irregular surfaces, and pelvic abnormality is seen (Fig. 3b). A striking radiographic feature ofAsl-s - / - mice are persisting growth plates at distal ulnar and radial and proximal tibial epiphyses. Persisting growth plates are also prominent in tail vertebrae at 4 and even 8 months of age, while in control animals growth plates are almost completely closed (Fig. 3b and data not shown). Together these radiological findings point at major alterations in bone formation. Pathologic Alterations in Bony and Cartilaginous Tissues. At the histological level prominent alterations of connective tissues are already observed in newbornAsl-s -/- mice. The cartilaginous anlagen of vertebrae (Fig. 4b) as well as skull bones exhibit a perturbed structure and an irregular surface and contain ballooned chondrocytes. At 4 weeks of age tracheal and articular cartilages are thickened and exhibit an irregular structure with ballooned, vacuolated chondrocytes FIG. 4. Histopathology of Asl-s -/- mice. (a) Median section (data not shown). Growth plates of long bones are highly through vertebrae of a newborn wild-type control mouse. Alcian blue broadened, the columnar arrangement of chondrocytes is lost, staining. (x50.) (b) Median section through vertebrae of a newborn and ballooned and vacuolated chondrocytes are not only found Asl-s -/- mouse. The architecture of the cartilaginous anlagen of in the opening zone as in wild-type controls but are demon- vertebrae is disturbed. Alcian blue staining. (X50.) (c) Growth plate of distal femur epiphysis of a 4-week-old wild-type control mouse. strable all across the longitudinal extension of growth plates Hematoxylin and eosin staining. (X50.) (d) Growth plate of distal (Fig. 4 c and d). femur epiphysis of a 4-week-old Asl-s -/- mouse. The growth plate Storage of Glycosaminoglycans. In all tissues investigated- is widened, the columnar arrangement of chondrocytes is lost, and liver, heart, lung, kidney, blood vessels, spleen, trachea, esoph- ballooned and vacuolated chondrocytes are found across the entire agus, stomach, small and large intestine, , salivary extension of the growth plate. Hematoxylin and eosin staining. (X50.) gland, testis, skin, adrenal gland, and lymph nodes-storage of (e) Liver section of a 4-month-old wild-type control mouse. Central glycosaminoglycans has been detected histologically and/or vein and arranged along sinus are shown. Mowry staining. ultrastructurally. Storage is most prominent in liver (Figs. 4f (x 100.) (f) Liver section of 4-month-old Asl-s - / - mouse. Sinus and heart 4h and lining cells are storing large amounts of glycosaminoglycans (blue). Sc), lung (Fig. Sb), (Figs. Sa), kidney (Fig. Sd), Mowry staining. (x 100.) (g) Myocard section of a 4-month-old wild- and skin (data not shown) and is detectable in these tissues type control mouse. Mowry staining. (X50.) (h) Myocard section of a almost exclusively in interstitial, fibroblast-like cells (Figs. 4h, 4-month-old AsJ-s -/- mouse. Storage of glycosaminoglycans in and 5 a and d), macrophages [e.g., Kupffer cells in liver interstitial cells. Mowry staining. (X50.) sinusoids (Fig. 4f)], and liver sinus endothelial cells (Fig. Sc). Parenchymal cells only rarely contain storage material [e.g., DISCUSSION parietal epithelial cells of renal Bowman's capsule (Fig. 5d) and liver parenchymal cells (data not shown) from an 8-month- A deficiency of the lysosomal enzyme ASB underlies the old affected animal]. With the exception of interstitial tissue of lysosomal storage disease MPS VI (Maroteaux-Lamy syn- the leptomeninges and of the choroid plexus and in older drome). To generate a mouse model for this disease the ORF animals (8-10 months) also in endothelial cells of blood of the ASB gene has been disrupted by homologous recom- vessels, no storage of glycosaminoglycans has been observed in bination. Mice homozygous for the mutant gene, which neither brain of animals up to 10 months of age (data not shown). express specific mRNA nor ASB activity, develop symptoms resembling the syndrome in humans and in the cat and rat Table 1. Length of long bones in MPS VI mice animal models of this disease described earlier (10, 11). Normal controls MPS VI-mice Urinary secretion of dermatan sulfate and granular inclusion bodies in leukocytes, which have served as diagnostic param- Bone n Mean + SD n Mean ± SD eters in human MPS VI (1, 22), are common to all three MPS Femur 7 1.64 ± 0.06 8 1.39 ± 0.09 VI animal models, including the MPS VI mouse described here Humerus 8 1.25 + 0.05 9 1.12 ± 0.06 (10, 11, 23). The symptoms due to alterations of connective Lengths (cm) are expressed as the mean ± SD. Differences are tissues-growth retardation, facial dysmorphia, and dysostosis statistically significant (P ' 0.001); n, number of bones investigated. multiplex with malformation of skull, vertebrae, ribs, pelvis, Downloaded by guest on September 28, 2021 8218 Biochemistry: Evers et al. Proc. Natl. Acad. Sci. USA 93 (1996) MPS VI cat (23) nor in the MPS VI mouse described here exhibit storage of glycosaminoglycans indicating that, as in human MPS VI, there is no primary central nervous system involvement. Even though all major pathological criteria of human MPS VI are detectable in the three animal models, these appear to be gradually attenuated from larger to smaller species. The link between the primary defect in MPS VI and the pathogenesis of the alterations in connective tissues (e.g. bones, cartilage, blood vessel walls, and cardiac valves) is not understood at present. The causality between the intracel- lular storage of dermatan sulfate in enlarged lysosomes and the thickening of bones, ligaments, and cardiac valves is not clear. Generation of a small animal model for MPS VI now allows for systematic investigations of composition and turnover of the extracellular matrix (e.g., collagens and ) of these tissues. In addition, the expression pattern of the respective genes may be studied in the affected tissues. Furthermore, the MPS VI mouse model provides an ideal tool for establishing new therapeutic approaches for this disease-e.g., enzyme substitution therapy and bone marrow, fibroblast, or myoblast implantation after in vitro retroviral gene transfer. This MPS VI model represents the second mouse model of an MPS storage disorder. MPS VII, which resulted from a spontaneous mutation (24), provided the first example, and this has proven to be an extremely valuable model for studying FIG. 5. Electron micrographs of storing cells fromAsl-s -/- mice. the effects of bone marrow transplants and enzyme replace- Membrane-bound storage vacuoles appear electron lucent partly with ment on this lysosomal storage disorder (25-27). The mouse condensed electron dense structures in electron micrographs of model of MPS VI resembles that of MPS VII in that it has 8-month-oldAsl-s -/- mice. (Bars = 2 ,Lm.) (a) I, interstitial cell in skeletal manifestations, but it differs in that it lacks the central myocard with large and smaller storage vacuoles; M, myocard cells; C, capillary lumen. (b) M0, tissue macrophage in the lung with multiple nervous system involvement of MPS VII. Although there are storage vacuoles; L, alveolar lumen; C, capillary lumen. (c) Liver also rat and cat models of MPS VI (11, 10), which are useful sinusoid with storage vacuoles in sinus lining endothelial cells (E), for some purposes, the small model has considerable advan- (H), and capillary lumen (C). (d) Bowman's capsule with tage over the larger animals in terms of life span, ease of storage vacuoles in parietal epithelial cells (pE) and interstitial cell (I). breeding, and control of the genetic background on which the BM, basal membrane of Bowman's capsule; P, podocyte. mutation is maintained. and long bones-which have been described for MPS VI N. patients (1) as well as for the cat (10, 23) and the rat (11) We thank Hartelt for excellent technical assistance, E. L. Punonnen for initial help with electron microscopy, S. Fedkenhauer model, are also present in the MPS VI mouse. Incomplete and B. Horchelhahn for help in the animal house, K. Rajewski (Koln, enchondral ossification due to aberrant growth of cartilaginous Germany) for providing the E-14-1 cell line, J. J. Hopwood (Adelaide, tissues may be a major cause for these alterations. Enlargement Australia) for providing the ASB substrate, H. Kresse (Munster, of liver and spleen have only been found in human MPS VI but Germany) for determinations, H.-C. Pauly for not in any of the animal models, even though inclusion bodies photographs, 0. Schunck and K. Nebendahl for veterinarian advice, have been seen preferentially in sinus lining cells of the liver and V. Gieselmann and K. Denzer for discussions. M.E. was supported in all three animal models (1, 10, 23). by a fellowship of the Boehringer Ingelheim Fonds. This work was Cardiac involvement with valvular dysfunction is a symptom, supported by the Deutsche Forschungsgemeinschaft (Grant Pe 310/ which has also been described for human MPS VI (1). In the 2-3) and the Fonds der Chemischen Industrie. MPS VI mouse, interstitial cells of cardiac muscle and blood vessel walls contain storage vesicles. Furthermore, cardiac 1. Neufeld, E. F. & Muenzer J. (1995) in The Metabolic and valves were found to be thickened due to interstitial edema in Molecular Bases ofInherited Disease, eds. Scriver, C. R., Beaudet, affected mice. Connective tissue cells in the cardiac valves A. L., Sly, W. S. & Valle, D. (McGraw-Hill, New York), Vol. 2, exhibited large, foamy cytoplasmic alterations (data not pp. 2465-2494. shown). Corneal opacity is a symptom of human MPS VI, 2. Matalon, R., Arbogast, B. & Dorfman, A. (1974) Biochem. Biophys. Res. Commun. 61, 1450-1457. which can be detected by split lamp investigation before it 3. O'Brien, J. S., Cantz, M. & Spranger, J. (1974) Biochem. Biophys. becomes visible upon inspection and causes serious visual Res. Commun. 60, 1170-1177. impairment (1). This alteration is also overt in the MPS VI cat 4. Peters, C., Schmidt, B., Rommerskirch, W., Rupp, K., Zuhlsdorf, (10, 23), whereas alterations of the cornea in the rat model (11) M., Vingron, M., Meyer, H. 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A. & Woo, L. C. (1994) Trends Genet. 10, 253-257. served in the smaller animal models rat (11) and mouse. 10. Jezyk, P. F., Haskins, M. E., Patterson, D. F., Mellman, W. J. & Neuronal cells of the central nervous system neither in the Greenstein, M. (1977) Science 198, 834-836. Downloaded by guest on September 28, 2021 Biochemistry: Evers et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8219

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