Defective proteoglycan sulfation of the growth plate zones causes reduced chondrocyte proliferation via an altered Indian hedgehog signalling

Matrix Biology 29 (2010) 453–460

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Matrix Biology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m a t b i o

Defective proteoglycan sulfation of the growth plate zones causes reduced chondrocyte proliferation via an altered Indian hedgehog signalling Benedetta Gualeni a,1, Marcella Facchini a,1, Fabio De Leonardis a, Ruggero Tenni a, Giuseppe Cetta a, Manuela Viola b, Alberto Passi b, Andrea Superti-Furga c, Antonella Forlino a, Antonio Rossi a,⁎ a b c

Department of Biochemistry “Alessandro Castellani”, University of Pavia, I-27100 Pavia, Italy Department of Experimental and Clinical Biomedical Sciences, University of Insubria, I-21100 Varese, Italy Centre for Pediatrics and Adolescent Medicine, Freiburg University Hospital, D-79106, Freiburg, Germany

a r t i c l e

i n f o

Article history: Received 30 December 2009 Received in revised form 18 April 2010 Accepted 3 May 2010 Keywords: Growth plate Diastrophic dysplasia Proteoglycan Chondrocyte proliferation Indian hedgehog Chondrodysplasia

a b s t r a c t Mutations in the sulfate transporter gene, SCL26A2, lead to cartilage proteoglycan undersulfation resulting in chondrodysplasia in humans; the phenotype is mirrored in the diastrophic dysplasia (dtd) mouse. It remains unclear whether bone shortening and deformities are caused solely by changes in the cartilage matrix, or whether chondroitin sulfate proteoglycan undersulfation affects also signalling pathways involved in cell proliferation and differentiation. Therefore we studied macromolecular sulfation in the different zones of the dtd mouse growth plate and these data were related to growth plate histomorphometry and proliferation analysis. A 2-fold increase of non-sulfated disaccharide in dtd animals compared to wild-type littermates in the resting, proliferative and hypertrophic zones was detected indicating proteoglycan undersulfation; among the three zones the highest level of undersulfation was in the resting zone. The relative height of the hypertrophic zone and the average number of cells per column in the proliferative and hypertrophic zones were significantly reduced compared to wild-types; however the total height of the growth plate was within normal values. The chondrocyte proliferation rate, measured by bromodeoxyuridine labelling, was also significantly reduced in mutant mice. Immunohistochemistry combined with expression data of the dtd growth plate demonstrated that the sulfation defect alters the distribution pattern, but not expression, of Indian hedgehog, a long range morphogen required for chondrocyte proliferation and differentiation. These data suggest that in dtd mice proteoglycan undersulfation causes reduced chondrocyte proliferation in the proliferative zone via the Indian hedgehog pathway, therefore contributing to reduced long bone growth. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The growth plate is a highly specialized cartilage structure, located between the primary and secondary centres of ossification, responsible for longitudinal growth of long bones. Based on morphological and functional characteristics, the growth plate can be divided in three major zones: the resting, proliferative and hypertrophic zones. Chondrocytes in the resting zone are rounded and sparsely distributed; and chondrocytes in the proliferative zone are flattened and Abbreviations: AMAC, 2-aminoacridone; bm, brachymorphic; BrdU, 5-bromo-2′deoxyuridine; CSPG, chondroitin sulfate proteglycan; DTD, diastrophic dysplasia; ΔDi0S, 3-O-β-(D-gluc-4-ene-uronosyl)-N-acetylgalactosamine; ΔDi-4S, ΔDi-6S, derivatives of ΔDi-0S with a sulfate at the 4 or 6 position of the hexosamine moiety, respectively; ECM, extracellular matrix; Ihh, Indian hedgehog; PAPS, 3′-phosphoadenosine-5′-phosphosulfate; P, post-natal day; TGF-β, transforming growth factor β. ⁎ Corresponding author. Dipartimento di Biochimica “Alessandro Castellani”, Via Taramelli, 3/B, I-27100 Pavia, Italy. Tel.: +39 0382 987229; fax: +39 0382 423108. E-mail address: [email protected] (A. Rossi). 1 These authors contributed equally to this work. 0945-053X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.matbio.2010.05.001

arranged in ordered columns separated by longitudinal septa. In the hypertrophic zone cells increase their metabolic activity causing cell enlargement as much as 10-fold compared to proliferating chondrocytes (Hunziker et al., 1987; Iannotti, 1990). Calcification and chondrocyte apoptosis commence at the bottom of the hypertrophic zone. The calcified cartilage matrix is essential for new bone formation as it provides the template for the synthesis of the primary spongiosa in the methapysis. Osteoblasts and osteoclasts remodel the bony trabeculae in the primary spongiosa into the mature secondary spongiosa (Gehron-Robey et al., 1992). Although chondrocytes in the growth plate zones are well characterized histomorphologically, a full knowledge of the signalling factors and cartilage extracellular matrix (ECM) components regulating growth plate chondrocyte differentiation and maturation in normal and pathological conditions is far from complete. Endochondral bone formation is tightly regulated by several molecules including systemic factors (growth hormone and thyroid hormone) and local soluble factors including Indian hedgehog (Ihh), parathyroid hormone related peptide, fibroblast growth factors, members of the

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transforming growth factor β (TGF-β) family, bone morphogenetic proteins and others (Karsenty and Wagner, 2002). The ECM of the growth plate is composed of an organized network of macromolecules which largely consists of collagens, the cartilage specific chondroitin sulfate proteoglycan (CSPG) aggrecan, hyaluronan and other proteins present in smaller amounts such as cartilage matrix protein, COMP, matrilins etc. Changes in the morphology and differentiation of growth plate chondrocytes parallel the changes in the composition of the ECM. The main ECM modifications include a decreasing gradient of type II and IX collagens from the resting zone to the lower hypertrophic zone (Mwale et al., 2002), while type X collagen is expressed exclusively in the hypertrophic zone (Sandell et al., 1994). Although the concentration of aggrecan (aggrecan per ECM volume) increases in hypertrophic cartilage due to continuing aggrecan synthesis and a reduction in matrix volume, there is an overall net loss of aggrecan prior to calcification (Matsui et al., 1991). The crucial role of aggrecan in endochondral ossification is demonstrated in the nanomelic (nm) chicken or in the cartilage matrix deficiency (cmd) mouse; in both strains a lack of aggrecan in the ECM results in a lethal form of chondrodysplasia with severe growth plate alterations (Schwartz and Domowicz, 2002). However more subtle defects in proteoglycan metabolism might affect growth plate performance, since it has been reported that the rates of aggrecan synthesis and degradation as well as its structural properties (mainly linked to the size and sulfation of glycosaminoglycan chains) are different in the zones of the ovine and bovine growth plate (Byers et al., 1997; Shapses et al., 1994). The key role of chondroitin sulfation for proper development, morphology and function of the growth plate has been demonstrated by the several chondrodysplastic mouse models with defects in the different steps of the sulfate activation pathway: the diastrophic dysplasia (dtd) mouse bearing a missense mutation in the Slc26a2 gene and resulting in reduced intracellular sulfate transport (Forlino et al., 2005), the brachymorphic (bm) mouse with a missense mutation in the 3′-phophoadenosine 5′-phosphosulfate (PAPS) synthetase 2 (Papss2) gene (Kurima et al., 1998), the C4st1 gene trap mutant for the chondroitin-4-sulfotransferase 1, which catalyses the sulfation at the 4-O position of chondroitin and dermatan sulfate (Kluppel et al., 2005) and the Jaws and the gPapp gene trap mutants both encoding a Golgi resident 3′-phophoadenosine 5′-phosphate 3′-phosphatase (Sohaskey et al., 2008; Frederick et al., 2008). In the neonatally lethal C4stgt/gt mouse and in the bm mouse CSPG undersulfation of the cartilage growth plate has been demonstrated in the whole epiphysis at post-natal days (P)1 and P6, respectively (Kluppel et al., 2005; Cortes et al., 2009). However since the growth plate consists of different zones with chondrocytes at different stages of differentiation, a detailed analysis of proteoglycan sulfation in the different zones would be necessary in order to link the morphological and functional defects of each zone to the structural defect of proteoglycans. Nowadays, microdissection protocols coupled to microanalytical techniques would allow the study of chondrocytes isolated from each zone of the growth plate from small size animals, such as mice; these studies have already been performed for transcriptomic analysis (Tsang et al., 2007; Wang et al., 2004; Belluoccio et al., 2008). In this work we have studied the growth plate in the dtd mouse, a knock-in mouse strain with cartilage proteoglycan undersulfation that reproduces several clinical, morphological and biochemical features of diastrophic dysplasia (DTD) in humans (Forlino et al., 2005). The phenotype is caused by a mutation in the Slc26a2 gene (also known as Dtdst) encoding for a widely distributed sulfate transporter (Hästbacka et al., 1994). Homozygous mutant mice are characterized by post-natal growth retardation, skeletal dysplasia and CSPG undersulfation of articular cartilage. The skeletal phenotype includes reduced toluidine blue staining of articular cartilage, chondrocytes of irregular size, delay in the formation of the secondary

ossification centre and early osteoporosis of long bones (Forlino et al., 2005). In order to characterize the biological relevance of chondroitin sulfation in growth plate morphology and function, we measured by disaccharide analysis CSPG sulfation of the resting, proliferative and hypertrophic zones of dtd and wild-type animals at P21; these data were related to growth plate histomorphometric parameters, to the cell proliferation rate and to the localization of Ihh, the most important long range morphogen for chondrocyte proliferation. 2. Results 2.1. Altered toluidine blue staining and aggrecan distribution in the ECM of dtd mice Sections of the proximal tibial epiphysis of wild-type and dtd mice at P21 were stained with toluidine blue (Fig. 1A). The ECM of the growth plate in mutant animals stained slightly less intensely than normal with this cationic dye, consistently with what was previously observed in the distal femoral epiphysis of dtd mice at other age points (Forlino et al., 2005). To determine whether reduced toluidine staining in mutant animals was due to proteoglycan undersulfation or reduced proteoglycan content in the growth plate ECM, we studied the distribution of aggrecan, the main cartilage ECM proteoglycan, in sections of the proximal tibial epiphysis of wild-type and dtd animals by immunohistochemistry with an antibody raised against the aggrecan core protein. Normal staining was observed in the cartilage matrix of dtd mice indicating that aggrecan distribution was within normal values (Fig. 1B). These data were confirmed by real time RT-PCR on RNA extracted from the microdissected growth plate (1.29 ± 0.45 vs. 1.36 ± 0.29 in dtd and wild-type mice, respectively). 2.2. Deficient sulfation of chondroitin sulfate chains in the different zones of the dtd mouse growth plate Preliminary sulfation studies by disaccharide analysis of the whole microdissected growth plate demonstrated an increase in the relative amount of non-sulfated disaccharide (ΔDi-0S) in dtd animals compared to the wild-types (21.1 ± 1.5% vs. 8.0 ± 0.9%, respectively; P b 0.0001, n = 5) confirming CSPG undersulfation previously observed in articular cartilage of SLC26A2 patients (Rossi et al., 1998) and of dtd mice as well (Forlino et al., 2005). To better characterize the sulfation defect at the growth plate level and in order to measure the extent of the defect in the morphologically and phenotypically distinct zones of the growth plate, 10 µm sagittal sections of the proximal tibia were microdissected in order to isolate the resting, proliferative and hypertrophic zones (Supplementary Figure S1). Each zone was then digested with chondroitinase ABC and ACII and released disaccharides labelled with 2-aminoacridone (AMAC); the three main chondroitin sulfate disaccharides (ΔDi-0S, ΔDi-4S and ΔDi-6S) were analysed and quantitated by reverse phase HPLC. In wild-type littermates significant differences (P b 0.05) in the relative amount of ΔDi-0S demonstrated that differences in sulfation were present among the zones considered: the relative amount of nonsulfated disaccharide was higher in the resting zone compared to the proliferative and hypertrophic zones. In the different zones of dtd animals a 2-fold, statistically significant, increase in the relative amount of non-sulfated disaccharide (ΔDi-0S) was observed when compared to wild-type mice (Fig. 2) indicating proteoglycan undersulfation. The highest level of CSPG undersulfation in dtd mice was observed in the resting zone (20.4 ± 2.9% vs. 8.2 ± 1.0% in the wildtype) and the degree of proteoglycan undersulfation decreased in the proliferative and hypertrophic zones, even if it was 2-fold higher compared to corresponding zones in wild-type littermates. The relative increase of the ΔDi-0S was related to an equivalent decrease

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Fig. 1. Toluidine blue staining and aggrecan detection in the proximal tibia growth plate of wild-type (wt) and mutant (dtd) mice at P21. (A) The cartilage matrix of dtd mice stains less intensely than normal with toluidine blue due to proteoglycan undersulfation. The resting (r), proliferative (p) and hypertrophic (h) zones are indicated. (B) Aggrecan immunohistochemistry shows a normal amount of the major cartilage proteoglycan in the ECM of dtd mice. Scale bar: 200 µm.

of the ΔDi-4S, while the relative amount of ΔDi-6S, that was extremely low (about 2%), was not affected by the sulfation defect.

P b 0.0001) and in the hypertrophic zones (5.4 ± 0.2 vs. 7.8 ± 0.3; P b 0.0001).

2.3. Altered histomorphometric parameters in the dtd growth plate

2.4. Chondrocyte proliferation in the growth plate of mutant animals is altered at P21

Sections stained with toluidine blue were used also for morphometric measurements and cell counting in the growth plate of P21 mice. In mutant mice the overall growth plate architecture was preserved, with the resting, proliferative and hypertrophic zone well delineated; in addition, the columnar alignment of proliferative and hypertrophic chondrocytes was maintained (Fig. 1A). Morphometric measurements were performed in wild-type and dtd mice in order to determine the total height of the growth plate and the relative extension of each zone (Table 1). In both strains the height of the growth plate decreased with age from P7 to P21 due to the expansion of the secondary ossification centre, while the relative extension of the proliferative and hypertrophic zones increased with age (data not shown). No significant differences were observed in the total growth plate height at P21, but the relative extension of the hypertrophic zone of the dtd mouse growth plate was significantly reduced when compared to wild-type littermates (27.2 ± 1.7% and 34.6 ± 2.1%, respectively; P b 0.05). We also evaluated the number of columns in a standardized area and the number of cells per column both in the proliferative and in the hypertrophic zones of wild-type and dtd mice (Table 1). The number of columns was normal in the proliferative and hypertrophic zones of the dtd mouse growth plate, but the average number of cells per column was significantly reduced when compared to wild-type littermates, both in the proliferative (8.8 ± 0.2 vs. 11.1 ± 0.2;

To determine whether the reduced longitudinal bone growth observed in dtd mice was the consequence of reduced chondrocyte proliferation at the growth plate level, we assessed the percentage of cells undergoing de novo DNA synthesis in the growth plate of wildtype and mutant mice at P21. For this purpose animals were injected with BrdU 2 h before sacrifice; positive BrdU cells were detected in sections of the proximal tibia growth plate by immunohistochemistry. BrdU labelled cells were detected only in the proliferative zone of wild-type and mutant animals (Fig. 3A). The proliferation rate of dtd mice, measured on the basis of the percentage of labelled nuclear profiles with respect to the total nuclear profiles (labelled with BrdU or with haematoxylin), was significantly reduced in the proliferative zone of mutant animals when compared to wild-type littermates (13.3 ± 1.0% vs. 16.4 ± 1.0%, respectively; P b 0.0001) (Fig. 3B). 2.5. Ihh signalling is reduced in the growth plate of dtd mice at P21 Ihh is expressed in a narrow zone of pre-hypertrophic chondrocytes and is a major regulator of chondrocyte proliferation in the growth plate (St-Jacques et al., 1999; Long et al., 2001). To find out whether reduced chondrocyte proliferation might be linked to an alteration in the Ihh signalling pathway, growth plate sections of the tibia from dtd and wild-type littermates were stained with a

Fig. 2. Sulfation of chondroitin sulfate proteoglycans in the resting, proliferative and hypertrophic zones of the tibia growth plate at P21. Proteoglycan sulfation was measured on the basis of the relative content of the ΔDi-0S, ΔDi-4S and ΔDi-6S disaccharide after digestion with chondroitinase of microdissected zones from sections of the tibia growth plate. In the three zones from mutant animals (dtd) the relative amount of non-sulfated disaccharide (ΔDi-0S) is higher compared to wild-type (wt) littermates, providing direct evidence of reduced CSPG sulfation; the highest undersulfation is in the dtd resting zone, but also in the proliferative and hypertrophic zones of mutant animals the level of CSPG sulfation is significantly altered compared to wild-types littermates. The mean values ± SD (n = 5) are reported in the graph on top of each column; *P b 0.0001.

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Table 1 Histomorphometric parameters of the tibial growth plate in mutant (dtd) and wildtype (wt) littermates at P21. wt

dtd

Height of the total growth plate (µm) 298.0 ± 9.8 299.3 ± 11.4 Relative height of the resting zone (%) 16.7 ± 5.4 21.3 ± 1.4 Relative height of the proliferative zone (%) 51.5 ± 1.7 50.5 ± 2.7 Relative height of the hypertrophic zone (%) 34.6 ± 2.1 27.2 ± 1.7 * 4 2 11.8 ± 0.4 12.9 ± 0.5 Number of columns in a 4×10 µm area in the proliferative zone Number of cells per column in the proliferative zone 11.1 ± 0.2 8.8 ± 0.2 ** 10.7 ± 0.3 10.3 ± 0.2 Number of columns in a 4×104 µm2 area in the hypertrophic zone Number of cells per column in the hypertrophic zone 7.8 ± 0.3 5.4 ± 0.2 ** The values represent the means ± SEM (n = 6). ⁎P b 0.05; ⁎⁎P b 0.0001.

Gli3 was within normal values (1.66 ± 0.27 vs. 1.14 ± 0.10 in dtd and wild-type mice, respectively). The expression data together with IHC results suggested that Ihh in dtd mice is normally expressed, but its distribution in the ECM of the growth plates is abnormal. To further support this hypothesis chondrocytes in primary cultures at two different cell densities were incubated for 24 h with recombinant mouse Ihh and proliferation was measured in the last 4 h (Fig. 5). In dtd cells treated with Ihh there was an increase in the cell proliferation rate vs. untreated culture similar to wild-type chondrocytes indicating that dtd cells exposed to exogenous Ihh rescue the normal proliferation phenotype and thus defective proliferation in vivo was linked to its abnormal distribution in the ECM. 3. Discussion

polyclonal antibody raised against the mature secreted Ihh protein (Fig. 4). In wild-type mice a positive Ihh signal was clearly evident in the ECM of pre-hypertrophic chondrocytes and the protein was distributed in the ECM from the pre-hypertrophic to the resting zones. Conversely, in the dtd growth plate the level of Ihh signal was significantly reduced in the pre-hypertrophic zone compared to wild type mice and, consequently, faint staining with the Ihh antibody was observed in the dtd proliferative and resting zones. By contrast, no significant changes were observed in the expression level of Ihh as detected by real time PCR on total RNA from microdissected growth plate of P21 mice (1.35 ± 0.55 vs. 1.27 ± 0.30 in dtd and wild-type mice, respectively). The Ihh pathway was further studied by measuring the RNA level of its receptor patched (Ptch1), and of the transcription factors Gli1 and Gli3 by real time PCR on total RNA from the microdissected growth plates of P21 animals. Ptch1 was normally expressed in the growth plate of dtd mice (2.36 ± 1.10 vs. 2.17 ± 0.87 in dtd and wild-type mice, respectively) and the level of activation of the Ihh pathway measured by the ratio of Gli1 to

Human inherited skeletal disorders as well as mouse models with mutations in relevant genes involved in proteoglycan sulfation have provided insights into the roles of this post-translational modification in skeletal development and bone growth. Proteoglycan undersulfation has been demonstrated in articular cartilage of patients (Rossi et al., 1998) and in the whole epiphysis of mouse models between P1 and P7 when the secondary ossification centre is not formed yet (Kluppel et al., 2005; Cortes et al., 2009). New protocols and equipments for microdissection coupled to microanalytical approaches have allowed the isolation and analysis of cells from heterogeneous tissues. These strategies have already been used for expression analysis of the different zones of the mouse growth plate by microarray (Belluoccio et al., 2008; Wang et al., 2004) or proteomic studies (Belluoccio et al., 2006). For the first time we have applied the same strategy to the analysis of CSPG sulfation in the different zones of the murine growth plate in dtd and wild-type littermates. These data, together with histomorphometric and proliferation studies, have provided new insight on the molecular

Fig. 3. Cell proliferation in the tibial growth plate of wild-type (wt) and mutant (dtd) mice at P21 by BrdU in vivo labelling. (A) Light micrographs show the distribution of immunoreactive chondrocytes at sacrifice 2 h after intraperitoneal injection of BrdU. In both dtd and wild-type mice a distinct nuclear staining reaction is limited to the proliferative zone. Sections were counterstained with haematoxylin. The resting (r), proliferative (p) and hypertrophic (h) zones are indicated. Scale bar: 100 µm. (B) Sections stained as in (A) were used to establish chondrocyte proliferation; the proportion of BrdU-labelled nuclei was calculated by comparing the number of BrdU labelled nuclei with the total number of chondrocytes in the proliferative zone (i.e. haematoxylin labelled nuclei + BrdU labelled nuclei). Mutant mice at P21 show significantly a lower proliferation rate when compared to wild-type littermates; *P b 0.0001. The values represent the means ± SEM (n = 6).

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Fig. 4. Ihh signalling in the dtd and wild-type mouse growth plate. Immunostaining of the proximal tibia growth plate of dtd and wild-type mice at P21 for the mature secreted form of Ihh; sections were counterstained with nuclear fast red. In the wild-type (wt) a strong Ihh signal (blue) in the extracellular matrix of pre-hypertrophic chondrocytes is clearly evident (A) as well as, at higher magnification, the gradient throughout the matrix in the proliferative and resting zones (B). By contrast, a marked reduced staining for Ihh is present in the dtd (dtd) growth plate. The resting (r), proliferative (p) and hypertrophic (h) zones are indicated. Scale bar: 100 µm. For negative control slides of Ihh immunohistochemistry see Supplementary Fig. S2.

and cellular events that could account for reduced long bone growth in the dtd mouse. P21 mice were used since i) at about this age (P24–26) growth rate is maximal in mice (Wang et al., 2002; Vanky et al., 1998); and ii) at this age the formation of the secondary ossification centre is complete in dtd and wild-type mice as well, thus both growth plates are morphologically similar. In fact, we have previously demonstrated that in dtd animals there is a delay in the formation of the secondary ossification centre compared to wild-type animals; nevertheless the secondary ossification centre is completely formed in mutant animals at P21 as well as in wild-types (Forlino et al., 2005). CSPG sulfation analysis in the resting, proliferative and hypertrophic zones of wild-type littermates demonstrated sulfation differences among the zones corresponding to the structural changes of

Fig. 5. Proliferation in dtd and wild-type chondrocytes during incubation with recombinant mouse Ihh. Chondrocytes at two different cell densities were incubated with 2 µg/ml recombinant Ihh for 24 h and proliferation was measured in the last 4 h with the CellTiter 96© AQueous One Solution Assay (Promega). Results are reported as percentage of proliferation increase with respect to untreated cultures. The proliferation rate in dtd chondrocytes is normal when cells are incubated in vitro with Ihh. Each point was measured in triplicate and each assay was repeated in three independent experiments. The mean values ± SD are reported in the graph on top of each column.

proteoglycans (size and aggregating properties) described previously (Byers et al., 1997; Matsui et al., 1991). In the three growth plate zones of dtd animals a 2-fold increase of the relative amount of ΔDi-0S was observed compared to wild-type littermates, indicating that the sulfation defect affects the three zones of the growth plate. The highest value of undersulfation was in the resting zone, while the level of undersulfation decreases in the proliferative and hypertrophic zones. The increase in the relative amount of ΔDi-0S was related to a decrease to the same extent of ΔDi-4S, while the relative low amount of ΔDi-6S was not affected by the sulfation defect. Reduced sulfation was also demonstrated in sections of mutant mice tibiae by reduced staining of the ECM with toluidine blue; however, sulfation differences among the different zones observed by disaccharide analysis were not appreciated at the histological level. Morphometric analysis demonstrated that CSPG undersulfation in dtd mice does not affect the total height of the growth plate at P21, but at this age the relative height of the hypertrophic zone was significantly reduced in mutant animals compared to wild-type littermates. Thus the level of undersulfation detected in the dtd mouse does not markedly affect the morphology of the growth plate; higher extent of proteoglycan undersulfation is necessary to disturb the height of the growth plate like in the bm mouse where a higher level of CSPG undersulfation in rib cartilage at P25 (Wikstrom et al., 1985) or in epiphyseal cartilage at P6 (Cortes et al., 2009) has been detected. CSPG undersulfation of the dtd growth plate influence chondrocyte organization and performance: the average number of cells per column in the proliferative and hypertrophic zones was significantly reduced. The reduced number of cells in the hypertrophic zone may account for its reduced relative height, since in this zone the volume of the matrix decreases and at the same time the cell size increases (Hunziker et al., 1987); thus the cell number per column is more important for the height of the hypertrophic zone than in the proliferative zone. In vivo chondrocyte BrdU labelling demonstrated reduced chondrocyte proliferation rate in the proliferative zone of dtd mice compared to physiological values accounting for a reduced number of cells per column in the proliferative zone and consequently in the hypertrophic zone.

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The rate of cell division in the proliferative zone of the growth plate is under the control of Ihh signalling; moreover, in the fetal growth plate Ihh directly acts on differentiation of round periarticular (resting) chondrocytes into flat, column-forming columnar chondrocytes (Kobayashi et al., 2005). Expression studies by real time RT-PCR of Ihh, its receptor, Ptch1, the activator, Gli1, and the repressor, Gli3, were within normal values; however immunohistochemistry of the growth plate with an antibody for the secreted form of Ihh demonstrated very reduced levels of Ihh in the pre-hypertrophic and hypertrophic region of mutant animals compared to wild-types and therefore the distribution of this morphogen in the ECM of the proliferative and resting zones was under the limit of detection. Abnormal distribution and diffusion of Ihh in the ECM of the dtd mouse growth plate might cause decreased chondrocyte proliferation. This is further supported by the observation that when dtd chondrocytes are exposed to exogenous Ihh in vitro, thus excluding the contribution of the undersulfated ECM, cells proliferate normally. It has been recently demonstrated that Ihh binds aggrecan through its glycosaminoglycan chains; the extent of binding relies on the degree of sulfation of the oligosaccharide chains, being the lowest for chondroitin (Cortes et al., 2009). Thus, the sulfation defect detected in the different zones of the growth plate alters Ihh distribution along the extracellular matrix of the zones of the growth plate. In particular in the resting zone of mutant mice with the highest value of CSPG undersulfation, the reduced distribution of Ihh in concert with its reduced binding to glycosaminoglycans can delay the differentiation of resting chondrocytes into proliferating column forming chondrocytes. In conclusion based on these data we propose a mechanism contributing to reduced bone growth in the dtd mouse. CSPGs of the growth plate show a different extent of sulfation in the different zones being more undersulfated in dtd compared to wild-type mice. CSPG undersulfation influence Ihh secretion and diffusion in the ECM of the growth plate. The altered distribution of this morphogen causes reduced chondrocyte differentiation in the resting zone and reduced chondrocyte proliferation in the proliferative zone, resulting in reduced number of cells per column. Thus, the primary structural proteoglycan defect causes reduced chondrocyte proliferation in the proliferative zone via the Ihh pathway. This molecular mechanism could account for reduced long bone growth in P21 dtd animals when the growth plate is delimited by the diaphysis at one end and the well developed a secondary ossification centre at the other. A similar mechanism for reduced long bone growth has been proposed for the bm mouse at P6, while a different mechanism involving the TGF-β signalling has been suggested in the C4stgt/gt mutant at P1 (Cortes et al., 2009; Kluppel et al., 2005). Different mechanisms contributing to the skeletal phenotypes depending on age are described also in other mouse models not linked to proteoglycan sulfation: the growth defect in the Grg5 null mutant associated with reduced Ihh signalling occurs during the first 4–5 weeks of age, but most mice recover retarded growth later (Wang et al., 2002). Also in our dtd mouse strain different age-related pathological mechanisms involving the development of the primary and secondary ossification centres might contribute to the skeletal phenotype and will require further attention in the future to correlate the age related long bone growth retardation to the molecular and morphological alterations of the growth plate. 4. Experimental procedures 4.1. Animals The dtd mouse is a “knock-in” for a c1184t transition causing an A386V substitution in the eighth transmembrane domain of the Slc26a2, which strongly reduces the activity of the transporter. Homozygous mutant mice show a chondrodysplastic phenotype

that recapitulates essential aspects of human DTD (Forlino et al., 2005). In this study, wild-type and homozygous mutant mice with a C57Bl/6J × 129/SV background at P21 were used. Genotyping of tail DNA to distinguish mutant from wild-type progeny was carried out by PCR or Southern blotting. Animals were bred with free access to water and standard pelleted food. Care and use of mice for this study were in compliance with relevant animal welfare guidelines approved by the Animal Care and Use Committee of the University of Pavia. 4.2. Tissue preparation Wild-type and homozygous mutant mice at P21 (n = 6) were injected intraperitoneally with 100 mg/kg 5-bromo-2′-deoxyuridine (BrdU, Sigma) in phosphate buffer saline (PBS, Sigma) 2 h before sacrifice. Tibiae were then excised, cleaned from the surrounding soft tissue and fixed in 4% formaldehyde in PBS at 4 °C overnight. Specimens were then rinsed in PBS, demineralised for a week under constant stirring at room temperature in 0.5 M EDTA (free acid, Sigma), brought to pH 7.1 with ammonium hydroxide, dehydrated in graded alcohols, cleared in xylene and embedded in paraffin, according to standard procedures. Sections, 5 µm thick for histology and immunohistochemistry or 10 µm thick for microdissection, were cut parallel to the long axis of the tibia using a RM2265 microtome (Leica) and mounted on Superfrost Plus slides (Menzel-Glaser). 4.3. Manual microdissection of the growth plate zones and CSPG sulfation analysis Sections (10 µm thick) of the tibia were deparaffinised in xylene and immersed briefly in absolute ethanol. Based on cell morphology, regions corresponding to the resting, proliferative and hypertrophic zones were microdissected under an inverted microscope (Leica DM IL) with the MicroDissector (Eppendorf) provided with a microchisel activated by ultrasound and moved by a micromanipulator (TransferMan® NK2, Eppendorf) (Supplementary Figure S1). Each isolated zone was then hydrated with 5–10 µl MilliQ water and harvested from the glass slide with a pipet tip. About 30–40 sections of each zone from the same growth plate were pooled for sulfation studies. Hyaluronic acid was removed by digestion with 4 U of Streptomyces hyaluronidase (Seikagaku Corporation) in 20 mM sodium acetate, pH 6.0, 75 mM NaCl at 60 °C overnight. At the end of the digestion, samples were centrifuged at 16,000 × g in order to isolate the insoluble pellet containing the whole extracellular matrix (and thus also CSPGs) from the supernatant containing the digested products of hyaluronic acid. Preliminary analysis demonstrated that during the digestion process some material containing chondroitin sulfate was released in the supernatant. For this reason the supernantant was ultrafiltrered with 10 kDa cut-off membranes (Biomax Ultrafree-0.5 Centrifugal filter units, Millipore) to remove hyaluronic acid digested products from chondroitin sulfate material that was recovered from the retentate and pooled with the insoluble pellet. Samples were then lyophilized and digested with 30 mU chondroitinase ABC and 30 mU chondroitinase ACII (Seikagaku Corporation) in 0.1 M ammonium acetate, pH 7.35, at 37 °C o/n; at the end of the digestion insoluble material was removed by centrifugation and released disaccharides in the supernatant were lyophilized. Disaccharides were derivatized with AMAC (Molecular Probes) as described previously (Viola et al., 2006). Separation and analysis of labelled disaccharides were performed with an HPLC (Waters) coupled to a fluorescence detector (Multy λ Fluorescence Detector 2475, Waters, λex = 442 nm and λem = 520 nm). Chromatography was carried out using a reverse phase column (Prontosil 120-3-C18-ace-EPS 3.0, 4.6 × 150 mm, Bischoff) at room temperature equilibrated with 0.1 M ammonium acetate, pH 7.0. AMAC-derivatives of disaccharides were separated by gradient elution with 0.1 M ammonium acetate buffer, pH 7.0 (eluent A) and

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acetonitrile (eluent B) (Viola et al., 2006). Sample peaks were identified and quantified comparing the fluorescence spectra with standard disaccharides (ΔDi-0S, ΔDi-4S and ΔDi-6S, Seikagaku Corporation) labelled with AMAC. 4.4. Histology and immunohistochemistry To evaluate the extension of the different zones of the growth plate, the number of columns per zone and the number of cells per column, sections were stained with toluidine blue according to standard procedures. Briefly, a toluidine blue working solution was prepared by mixing 5 ml of 1% toluidine blue O (Sigma) in 70% ethanol and 45 ml of 1% sodium chloride in distilled water. Sections were deparaffinised and hydrated to distilled water, then stained with the toluidine blue working solution, dehydrated quickly, cleared in xylene and mounted. For aggrecan immunohistochemical detection, sections were deparaffinised, hydrated, pre-treated with 1 U/ml chondroitinase ABC (Siekagaku Corporation) to remove glycosaminoglycan chains and then incubated with an anti-aggrecan primary antibody (H-300, Santa Cruz Biotechnology), which was then detected with the HistoMouse™-MAX kit (Zymed Laboratories) according to the manufacturer's suggestions. Sections were counterstained with haematoxylin. In preparation for Ihh immunohistochemistry sections were digested with 1 mg/ml bovine testicular hyaluronidase (Sigma), incubated with anti-Ihh polyclonal antibody (R&D Systems, 1:15) and signal amplification and detection was performed using alkaline phosphatase tyramide signal amplification (PerkinElmer). Sections were counterstained with nuclear fast red. For immunohistochemical detection of proliferating cells, sections were deparaffinised, hydrated and treated with the BrdU Staining Kit (Zymed Laboratories), according to the manufacturer's suggestions; sections were counterstained with haematoxylin. 4.5. Chondrocyte proliferation assay Chondrocytes were released from epiphyseal cartilage of P1 mice by digestion with 1 mg/ml collagenase type II (Invitrogen) in DMEM at 37 °C overnight. Dissociated cells were plated in the same medium containing 10% heat-inactivated fetal calf serum (FCS). For proliferation studies induced by Ihh, primary cultured cells were seeded in 96 well plates at two different cell densities, 5 × 103 and 1 × 104 cells/ well, respectively, and incubated in MEM containing 25 µM cystine and methionine, 250 µM Na2SO4 and 10% heat-inactivated FCS at 37 °C in 5% CO2 for 24 h. Cells were then pre-incubated in the same MEM containing 0.5% heat-inactivated FCS at 37 °C for 4 h and then incubated with 2 µg/ml recombinant mouse Ihh (R&D Systems) in the same medium for 24 h at 37 °C in 5% CO2 (Shimo et al., 2004). Proliferation was measured by pulse labelling in the last 4 h of culture with the CellTiter 96© AQueous One Solution Assay (Promega) according to the manufacturer's protocol. Each point was measured in triplicate and each assay was repeated in three independent experiments. Cell proliferation in Ihh treated cells was measured as a relative increase with respect to cells incubated without Ihh.

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immediately suspended in RLT Plus buffer and total RNA was extracted using the RNeasy Plus Micro Kit (Qiagen) including the DNase digestion step according to the manufacturer's instructions. Quantitation and integrity of total RNA were determined by using the Agilent Bioanalyzer 2100 (Agilent). RNA (100 ng) was reverse transcribed using the High Capacity cDNA Archive Kit (Applied Biosystems) as per manufacturer's recommendation. Relative quantitative real time PCR experiments were performed using aggrecan (Mm00545794_m1), Ihh (Mm00439613_m1), Ptch1 (Mm00970977_m1), Gli1 (Mm00494645_m1) and Gli3 (Mm00492345_m1) Assays on Demand from Applied Biosystems and Gapdh (Mm99999915_g1) was used as housekeeping gene for expression normalization. Analysis was performed using the MX3000P (Stratagene). Each sample was run in triplicate in 96 well plate using 1× Universal Master Mix and 1× TaqMan specific probe (Applied Biosystems). Thermal cycling conditions included a pre-run of 10 min at 95 °C. Cycles conditions were 40 cycles at 95 °C for 15 s and 60 °C for 1 min according to the TaqMan Universal PCR protocol (Applied Biosystems). Three dtd and three wild-type mice were used; relative expression was determined with the ΔΔCt method. Data are expressed as mean± SEM; Student's t-test was applied and a P-value b0.05 was considered significant. 4.7. Measurements and statistical analyses Pictures of the stained sections were taken using a DFC480 digital camera (Leica) connected to a light microscope (Dialux 20, Leica). The resting, proliferative and hypertrophic zones were delineated on the basis of cell morphology, as reported elsewhere (Vanky et al., 1998). The height of the different zones of the growth plate, the number of columns per zone and the number of cells per column were measured on toluidine blue stained sections using the Leica Application Suite image analysis software (Leica). To determine the height of each zone, at least 20 vertical measurements per zone per section were performed. To determine the number of cells per column, at least 20 columns per zone per section were analysed. An average of 4 sections per animal was considered and measurements were performed centrally in the zones. For proliferation assays, all chondrocyte nuclei and BrdU-positive chondrocyte nuclei in the proliferative zone of the growth plate were counted in order to evaluate the percentage of replicating cells. We analysed an average of 6 sections per animal and measurements were performed centrally in the zones. Statistical differences between different groups were evaluated with ANOVA and Student's t-test using Sigma Plot Statistic 11.0 software; a P-value b 0.05 was considered significant. Acknowledgements We thank Dr. Fabio Pecora for kind help and support during this study. This work was supported by grants from Telethon-Italy (grant no. GGP06076), Fondazione Cariplo (Milan, Italy), Regione Lombardia (Project REGLOM16) and the European Community (FP6, “EuroGrow” project, LSHM-CT-2007-037471).

4.6. Growth plate RNA extraction and real time RT-PCR

Appendix A. Supplementary data

Tibiae were dissected from P21 mice and then molded in Tissue Tek O.C.T. compound and rapidly frozen in liquid nitrogen. Frozen longitudinal sections (10 µm) of the proximal tibial epiphyses were cut on a Leica CM1850 cryostat at −30 °C and mounted on Superfrost Plus slides. Slides were then fixed in 70% ethanol at −30 °C for 1 min, washed in DEPC treated water at room temperature for 3 min to remove OCT and then dehydrated in graded alcohols. The growth plate was then microdissected under an inverted microscope (Leica DM IL) with the MicroDissector (Eppendorf) and the tissue was

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