A mouse model of chondrocyte-specific somatic mutation reveals a role for Ext1 loss of heterozygosity in multiple hereditary exostoses

Kazu Matsumotoa, Fumitoshi Iriea, Susan Mackemb, and Yu Yamaguchia,1

aSanford Children’s Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037; and bCancer and Developmental Biology Laboratory, National Cancer Institute, Frederick, MD 21702

Edited* by Erkki Ruoslahti, University of California, Santa Barbara, CA, and approved May 12, 2010 (received for review December 23, 2009) Multiple hereditary exostoses (MHE) is one of the most common The current consensus is that the copolymerase activity resides skeletal dysplasias, exhibiting the formation of multiple cartilage- in the EXT1 , and that the association of the EXT2 capped bony protrusions () and characteristic protein with the EXT1 protein is necessary for the proper lo- bone deformities. Individuals with MHE carry heterozygous loss- calization of EXT1 in the Golgi apparatus (14, 15). Genetic of-function mutations in Ext1 or Ext2, which together encode ablation of either results in essentially complete abrogation an essential for synthesis. Despite the of HS production in cells and tissues (16, 17). The majority of identification of causative genes, the pathogenesis of MHE remains cases of MHE carry frameshift or missense mutations in EXT1 or unclear, especially with regard to whether osteochondroma results EXT2 (18, 19). from loss of heterozygosity of the Ext genes. Hampering elucida- Despite the unambiguous identification of causative genes and +/− +/− tion of the pathogenic mechanism of MHE, both Ext1 and Ext2 their function, the pathogenic mechanism of MHE remains heterozygous mutant mice, which mimic the genetic status of hu- elusive. It was originally hypothesized that EXT1 and EXT2 are man MHE, are highly resistant to osteochondroma formation, es- tumor suppressor genes, and the loss of heterozygosity (LOH) at pecially in long bones. To address these issues, we created a mouse these loci plays a key role in osteochondroma formation (8, 9). model in which Ext1 is stochastically inactivated in a chondrocyte- However, the data from genetic analysis of are specific manner. We show that these mice develop multiple osteo- equivocal (20–23). It is also uncertain whether osteochondroma chondromas and characteristic bone deformities in a pattern and is a true neoplasm, which by definition is derived from clonal a frequency that are almost identical to those of human MHE, sug- expansion of progenitor cells, or whether it represents a focal gesting a role for Ext1 LOH in MHE. Surprisingly, however, geno- developmental malformation (20). Further confounding the is- +/− +/− typing and fate mapping analyses reveal that chondrocytes sue is the fact that both Ext1 and Ext2 heterozygous mu- constituting osteochondromas are mixtures of mutant and wild- tant mice, which mimic the genetic conditions of human MHE type cells. Moreover, osteochondromas do not possess many typi- faithfully, develop osteochondromas only in rib bones with a low cal neoplastic properties. Together, our results suggest that inacti- penetrance (17, 24). Ext1 fi vation of in a small fraction of chondrocytes is suf cient for To address these unsolved issues, we created a mouse model the development of osteochondromas and other skeletal defects based on stochastic, tissue-specific inactivation of Ext1. We show associated with MHE. Because the observed osteochondromas in that mice with inactivation of Ext1 in a minor fraction of chon- our mouse model do not arise from clonal growth of chondrocytes, drocytes develop multiple osteochondromas and bone deform- they cannot be considered true neoplasms. ities in a pattern almost identical to human MHE, whereas mice carrying monoallelic inactivation do not, suggesting a role for bone development | heparan sulfate | osteochondroma LOH in the process. Surprisingly, we found that osteochondromas do not develop in these mice by clonal growth of mutant chon- ultiple hereditary exostoses (MHE) is an autosomal drocytes. Our results provide answers to long-standing questions Mdominant disorder characterized by the formation of concerning the pathogenic mechanism of this disorder, and fur- multiple cartilage-capped bony protrusions (osteochondroma). thermore, indicate that, despite the likely contribution of LOH in It is one of the most common skeletal dysplasias in humans, its initiation, osteochondroma in MHE is not a true neoplasm in with a prevalence of 1 in 18,000 (1, 2). In MHE, osteochon- its strictest sense. dromas occur in almost all types of bones, including flat bones, rib bones, and vertebrae, although they most typically form at Results the distal end of long bones. MHE patients also exhibit various To test the hypothesis that inactivation of Ext1 occurring in deformities of the skeletal system, such as short stature, limb a small fraction of chondrocytes is the pathogenic mechanism of length inequalities, bowing of the limb bones, and scoliosis (3– MHE, we used a method of stochastic inactivation of loxP- F 5). Malignant transformation into metastatic chondrosarcoma flanked Ext1 alleles (Ext1 ) using a tamoxifen-dependent Cre ERT is the most serious complication of MHE, but its frequency is transgene driven by the Col2a1 promoter (Col2-Cre ) (25). not particularly high (6). Genetic linkage analysis has identified We originally intended to control the level of recombination ERT two genes as being associated with the vast majority of MHE: using different doses of tamoxifen. Unexpectedly, Col2-Cre ; EXT1, located on 8q24.1, and EXT2, located on chromosome 11p11 (7–11). It has been established that EXT1 EXT2 and jointly encode a essential for Author contributions: Y.Y. designed research; K.M. and F.I. performed research; S.M. heparan sulfate (HS) synthesis (12, 13). contributed new reagents/analytic tools; K.M. and F.I. analyzed data; and Y.Y. wrote Heparan sulfate (HS) is a highly sulfated linear polysaccharide the paper. with a backbone of alternating N-acetylglucosamine (GlcNAc) The authors declare no conflict of interest. and glucuronic acid (GlcA) residues. The EXT1 and EXT2 pro- *This Direct Submission article had a prearranged editor. teins form an oligomeric complex that catalyzes the copoly- 1To whom correspondence should be addressed. E-mail: [email protected]. merization of GlcNAc and GlcA residues, thereby elongating the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. HS backbone. 1073/pnas.0914642107/-/DCSupplemental.

10932–10937 | PNAS | June 15, 2010 | vol. 107 | no. 24 www.pnas.org/cgi/doi/10.1073/pnas.0914642107 Downloaded by guest on September 24, 2021 F/F Ext1 mice developed multiple osteochondromas and other the radial head were observed in 91.7% of Ext1-SKO mice, where- MHE-like skeletal defects without tamoxifen treatment (Fig. 1). as scoliosis was found in 58.3% (Fig. 1 D and E, and Table S1). As described below, this turned out to be due to a low-level Together with the pattern of osteochondroma formation men- leakiness of the transgene, which causes random deletion of tioned above, these results show that Ext1-SKO mice phenocopy Ext1 in a small fraction of chondrocytes (see below). For con- the skeletal defects of MHE quite faithfully. In comparison, het- ERT F/F ERT F/+ venience, Col2-Cre ;Ext1 mice that are raised without ta- erozygous SKO mice (Col2-Cre ;Ext1 ) never developed moxifen treatment are called Ext1-SKO mice (“SKO” repre- osteochondromas or other skeletal defects (Table S1). The same is senting “stochastic/sporadic knockout”) in this article. true for another conditional heterozygous Ext1 mutant model in In Ext1-SKO mice, multiple bony protrusions involving the which Ext1 is monoallelically inactivated in the entire limb bud F/+ wrist, fibula, shoulder, and rib were identified by radiographic and mesenchyme (Prx1-Cre;Ext1 )(Table S1). These results strongly macroscopic observations (Fig. 1 A and B). Histological exami- suggest a requirement for biallelic inactivation of Ext1 in the de- nation of these lesions revealed bony tuberosities with a cartilage velopment of MHE-like phenotype. In addition to the develop- cap (Fig. 1C), which is consistent with the histological features of ment of osteochondromas, Ext1-SKO mice display mild abnormal- osteochondromas. Table S1 summarizes the occurrence of osteo- ities in the growth plate and joint cartilage (Fig. S1). ERT chondromas at different locations in Ext1-SKO mice at P28. The The Col2-Cre transgene used in this study has been repor- penetrance of the long bone exostosis phenotype (defined as the ted to exhibit a low-level leakiness (25). Thus, we hypothesized presence of at least one conspicuous osteochondroma in long that the phenotypes observed in Ext1-SKO mice are caused by Ext1 leaky Cre activity. To test this hypothesis, we analyzed the spa- bones) in -SKO mice was 100%. This is in stark contrast to the ERT observation that mice carrying the same genotype as human MHE tiotemporal recombination pattern of the Col2-Cre transgene +/− +/− patients (i.e., Ext1 and Ext2 ) never develop osteochon- using the Rosa26-lacZ reporter (26) in whole-mount skeleton dromas in long bones (17, 24) (Table S1). (Fig. S2) and sections through the growth plate (Fig. 2A). In the Individuals with MHE frequently exhibit skeletal defects other whole-mount forelimb at E14.5, a small number of lacZ+ clusters than osteochondromas, such as short stature, bowing deformity in developing limb skeleton were observed (Fig. S2, E14.5), in- of the forearm, subluxation/dislocation of the radius, and scoli- dicating that stochastic recombination occurs as early as E14.5. + osis. Ext1-SKO mice had short stature [wild type, 8.04 ± 0.13 cm Postnatally, a small number of lacZ clusters were found in the + (n = 16); Ext1-SKO, 5.57 ± 0.15 cm (n = 12); at P28]. The femoral head cartilage at P0, and the number of lacZ clusters bowing deformity of the radius and the subluxation/dislocation of showed no significant increase during the period from P0 to P28 (Fig. S2,P0–P28). In the growth plate, lacZ+ cells form columnar clusters, corresponding to the longitudinal growth of chondro- cytes (Fig. 2A). As is the case with the whole-mount analysis, the frequency of lacZ+ cells was low both in P7 and P28 growth plates. No lacZ+ cells were found in the perichondrium, including the area known as the groove of Ranvier (27). As an indepen- dent measure for recombination efficiency, we also performed an allele-specific PCR analysis (Fig. 2 B and C). Consistent with the ΔF specificity of the Col2a1 promoter, no recombined allele (Ext1 ) was detected in tissues other than cartilage (Fig. 2B). Quantitative PCR analysis revealed that 6% of the Ext1 allele is recombined in ERT F/F tibial cartilage of Col2-Cre ;Ext1 mice, whereas the ratio in rib cartilage was 15% (Fig. 2C). Together, these results demon- strate that in Ext1-SKO mice, Ext1 is stochastically inactivated in a minor fraction of chondrocytes. The development of osteochondromas was examined in the wrist joint area (Fig. 3), where osteochondromas develop at 100% penetrance. The first sign of abnormality was found at P14, when the width of the distal radius and ulna became increased and cartilage was formed in abnormal locations (Fig. 3A, arrowheads in P14). At P21, the overgrowth and deformity of cartilage pro- gressed (arrowheads in P21), and at the same time, small carti- laginous islands were formed in the epiphysis (arrows in P21). At P28, multiple protrusions that closely resemble osteochondromas in MHE were established (arrows in P28). Histologically, micro- scopic exostoses could be detected at P14 (Fig. 3B, arrows in P14), as well as the overgrowth of cartilage tissues between the radius and ulna (arrowheads in P14). Similar microscopic osteochon- dromas were also seen in the distal femur at P14 (Fig. 3C). In rib bones, the development of osteochondromas occurs during the period of P7 to P28, slightly earlier than in long bones (Fig. S3). These results demonstrate that osteochondromas develop quickly Fig. 1. Skeletal phenotype of Ext1-SKO mice. (A) X-ray images of P28 Ext1- during the early postnatal period corresponding to rapid bone SKO mice show multiple bony protrusions (arrows) in the long bones and the growth, rather than at the time of gene ablation or the onset of HS ERT rib cage. (B) Macroscopic views of bony protrusions in the wrist joint and the expression. The Col2-Cre -dependent recombination commen- rib cage of P28 Ext1-SKO mice (arrows). (C) Safranin O/Fast Green-stained ces as early as E14.5 and does not change during early postnatal sections of exostosis-like lesions developed in P28 Ext1-SKO mice. Note that these bony protrusions have a cartilage cap with the growth plate-like tissue period (see above), and HS is continuously expressed in de- structure, reminiscent to osteochondromas in human MHE. (D and E) Ext1- veloping limbs from the early limb bud stage onwards (28, 29).

SKO mice mimic skeletal deformities seen in human MHE. (D) Bowing of the To gain insight into the mechanisms of osteochondroma de- BIOLOGY

radius (arrow) and the subluxation/dislocation of its head (arrowheads). (E) velopment, histological and molecular properties of osteochon- DEVELOPMENTAL Scoliosis of the thoracic vertebral column (arrowheads). dromas were examined. Safranin O/Fast Green staining showed

Matsumoto et al. PNAS | June 15, 2010 | vol. 107 | no. 24 | 10933 Downloaded by guest on September 24, 2021 Fig. 2. Col2-CreERT transgene without induction with tamoxifen drives stochastic recombination of floxed alleles in a small fraction of chondrocytes. (A) Time course of Col2-CreERT-mediated recombination under noninduced conditions. R26R;Col2-CreERT mice were raised without tamoxifen treatment, and sections through the growth plate of the femur and tibia were stained with X-gal at indicated time points. Note that the frequency of lacZ+ cells is low, and that lacZ+ cells form clusters corresponding to the columnar organization of chondrocytes in the growth plate. See also Fig. S2 for a similar analysis in whole-mount skeletons. (B) Tissue specificity of Ext1 recombination driven by Col2-CreERT without tamoxifen treatment. Genomic DNA from indicated tissues of P28 Ext1- SKO mice was analyzed by PCR specific for the recombined Ext1ΔF allele. (C) Quantitative PCR analysis of recombination efficiency. The amounts of the Δ Δ recombined Ext1 F allele and intact Ext1F allele were determined by quantitative PCR and represented as the percentage of Ext1 F alleles (black bars) to total Ext1 alleles. Genomic DNA samples from proximal tibial cartilage and rib cartilage of P28 Ext1-SKO mice were examined. The results from two quantitative Δ − controls (kidney DNA from Ext1-SKO mice, which should contain no Ext1 F allele, and brain DNA from Ext1+/ heterozygous mice, which is expected to give a 1:1 ratio) verify approximate linearity of the assay. All results are based on duplicate samples from three independent mice in each genotype.

ΔF F that osteochondromas in Ext1-SKO mice have the growth plate- ratio between the recombined Ext1 and intact Ext1 alleles was ΔF like cellular architecture as seen in human MHE (SafO/FG in variable, and in most samples examined, the Ext1 allele repre- Fig. 4 A and B). Expression domains of Col2a1, Ihh, and Col10 sented a minor proportion of the total Ext1 alleles present. To are organized rather orderly as in the normal growth plate (Fig. further define the genetic makeup of osteochondromas, we per- ERT F/F 4A). Moreover, the frequency of replicating cells in osteochon- formed a fate mapping analysis using R26R;Col2a1-Cre ;Ext1 dromas is not significantly different from that in the normal compound mice and determined the contribution of Ext1-null growth plate (Fig. 4B). These results suggest that osteochon- chondrocytes to osteochondromas. We found that the cartilage cap dromas in Ext1-SKO mice do not possess many typical neoplastic of osteochondromas contained variable numbers of lacZ+ chon- properties, such as disorganized and unregulated growth; rather, drocytes, but were never comprised 100% of the lacZ+ cells (Fig. they look more like ectopic growth plates. 5B), which is consistent with the result from the genotyping anal- The fact that the inactivation of Ext1 in a small fraction of ysis. Together, these results demonstrate that osteochondromas chondrocytes leads to the formation of multiple osteochondromas are not simple clonal growth of Ext1-null chondrocytes. led us to examine whether osteochondromas are clonal growth of Ext1-null chondrocytes. To address this issue, we first analyzed Discussion DNA samples isolated from the cartilage cap region of osteo- In this article, we show that Ext1-SKO mice recapitulate many of chondromas by laser capture microdissection (LCM) (Fig. 5A the key features of human MHE. Most importantly, this mouse Upper). Because the amount and quality of DNA that could be model develops multiple osteochondromas in long bones, which is obtained from fixed osteochondroma tissues were not sufficient the hallmark of MHE and which could not be reproduced pre- for the quantitative PCR protocol, we performed semiquanti- viously in Ext1 or Ext2 heterozygous mutant mice (17, 24). It has tative analysis using the standard allele-specific PCR protocol been debated that whether LOH plays a role in the pathogenesis of (Materials and Methods). Surprisingly, this analysis revealed that MHE (20–23). Our present findings lend strong support to the all 15 osteochondromas examined were heterogeneous, contain- LOH hypothesis regarding the mechanism of osteochondroma ing both the recombined and intact alleles (Fig. 5ALower). The formation. Although biallelic inactivation of Ext1 in individual

10934 | www.pnas.org/cgi/doi/10.1073/pnas.0914642107 Matsumoto et al. Downloaded by guest on September 24, 2021 Fig. 4. Histological characterization of osteochondromas in Ext1-SKO mice. (A) Analysis of the tissue organization of osteochondromas by in situ hy- bridization for Col2a1, Ihh, and Col10. Adjacent sections were stained with Safranin O and Fast Green (SafO/FG). (B) Analysis of cell proliferation. Sec- tions of osteochondromas were double-stained with anti-phosphorylated histone H3 (pHH3) and DAPI. Quantitative analysis of pHH3+ cells (bar graph) shows that there is no significant difference in the frequency of replicating cells between the cartilage cap region of osteochondromas and the normal growth plate. GP, growth plate; OC, osteochondroma.

This study has provided insight into the cellular origin of osteochondroma. It has been reported that transgenic Col2a1 promoters tend to be expressed not only in chondrocytes but also Fig. 3. Development of osteochondromas in Ext1-SKO mice. (A) Whole- in early perichondrial progenitors (30). However, activation of ERT mount skeletal preparations in the wrist joint area. (B) Sections through the the Col2a1-Cre transgene used in this study in perichondrial wrist joint area. (C) Sections through the knee joint area. Note that at P7, no cells does not occur unless the transgene is induced with tamox- apparent abnormalities are observed in Ext1-SKO mice either by macroscopic fi ifen before E13 when chondrogenic and perichondrial precursors (A) or microscopic (B and C) analysis. The rst detectable abnormalities in are not yet segregated (25). Indeed, we have never observed the wrist joint area are the overgrowth of the growth plate cartilage + R26R;Col2-CreERT (arrowheads in A and B) and the formation of microscopic exostoses (arrows lacZ labeling in the perichondrium of mice in B) at P14. These abnormalities become more prominent at P21, and by P28, (see Results). Thus, osteochondromas in Ext1-SKO mice are likely multiple bony protrusions with a cartilage cap are formed in the wrist joint to originate from chondrocytes in the growth plate. It should be area (A). Osteochondroma formation in the knee joint area takes a similar noted, however, that this conclusion does not mutually exclude time course (C). the possibility that perichondrial cells can also be an origin of osteochondromas. This latter possibility will need to be examined by studies using different Cre systems. + fi fi lacZ cells could not be speci cally veri ed in our model, its likely Although our results represent a significant development to- importance in osteochondroma formation is further suggested ward understanding the pathogenesis of MHE, there are a number by the total absence of osteochondromas in two control mutant of remaining questions. For instance, how does the inactivation of — Col2-CreERT;Ext1F/+ Prx1-Cre;Ext1F/+— mice namely, and in Ext1 in a small fraction of chondrocytes result in the formation of which biallelic inactivation cannot occur (Table S1). osteochondromas, which we now know are nonclonal? Our data It is surprising that osteochondromas that developed in Ext1- suggest that the presence of Ext1 null chondrocytes is required for SKO mice invariably contain both Ext1 null and wild-type chon- the initiation of osteochondromas in these mice, but the sub- drocytes. Moreover, wild-type cells constitute a major population sequent growth of osteochondromas is not directly due to the of chondrocytes in most of the osteochondromas examined. Thus, unregulated proliferation of mutant cells. In other words, despite it is unlikely that wild-type cells are just an incidental component the role of LOH in its development, the pathogenesis of MHE is of osteochondromas. This heterogeneous composition of osteo- probably not fully explained by the classical two-hit model. An chondromas may be a part of the reason why the previous studies interesting possibility, although speculative at this point, is that with human osteochondroma specimens have generated somewhat mutant chondrocytes lacking HS exert some sort of cell non- conflicting views concerning LOH as the genetic mechanism of autonomous effect on wild-type chondrocytes. Another key ques- MHE (20–23). Together with the nearly normal expression pattern tion is whether the same genetic mechanism as we found in this of differentiation markers and the level of cell proliferation similar study applies to human MHE. Considering the present findings

to that in the normal growth plate (Fig. 4), osteochondroma de- regarding the importance of biallelic inactivation, it should be of BIOLOGY Ext1

veloped in -SKO mice resembles an ectopic growth plate more interest to revisit the issue of LOH in human osteochondromas. If DEVELOPMENTAL than a neoplasm. LOH does indeed underlie human MHE, it is then important to

Matsumoto et al. PNAS | June 15, 2010 | vol. 107 | no. 24 | 10935 Downloaded by guest on September 24, 2021 Fig. 5. Chondrocytes comprising osteochondromas are mixtures of mutant and wild-type cells. (A) Analysis of the genotype of chondrocytes in osteo- chondromas by laser-capture microdissection (LCM). Cartilage cap regions of osteochondromas were isolated by LCM, and genomic DNA specimens from these tissue fragments were analyzed by allele-specific PCR. (Upper Left) Representative result of LCM. (Upper Right) Result of the PCR analysis of DNA from − − nine independent osteochondromas. As controls, DNA from cartilage of Ext1+/ mice (Ext1+/ ) and that from cartilage of Ext1F/F mice (Ext1F/F) were also analyzed. Note that all osteochondromas tested contain both the recombined Ext1ΔF and intact Ext1F alleles. (Lower) Ratio of the Ext1ΔF and Ext1F alleles estimated by semiquantitative PCR in the 15 osteochondromas developed in the femur, radius, and ulna. Note that the percentage of the Ext1ΔF allele is highly variable, ranging from 9.6% (#14) to 52.6% (#1). (B) Fate mapping analysis of chondrocytes comprising osteochondromas. To evaluate the contribution of Ext1 null chondrocytes to osteochondromas, Col2-CreERT;Ext1F/F mice carrying the R26R reporter gene were bred, and osteochondromas developed in these mice were stained with X-gal to identify cells that had undergone Cre-mediated recombination. A gallery of four representative results is shown at lowand high magnifications. Osteochondromas indicated by arrowheads in Left are shown in Right at high magnification. Note that none of the osteochondromas examined were entirely composed of cells that undergone recombination. The contribution of recombined cells in osteochondromas is highly variable.

determine how LOH occurs in human chondrocytes. Alternatively, Analysis of the Skeleton. For whole-mount analysis of skeletons, mice were it is possible that mouse and human chondrocytes do not respond eviscerated and fixed in 95% ethanol overnight. The preparations were – in the identical manner to heterozygous levels of Ext1, in such stained with alcian blue for 1 3 d, rinsed in 95% ethanol, incubated 2% KOH for 1–24 h, and stained with alizarin red for 1–3 d. The stained preparations a manner that mouse cells tolerate heterozygous levels of Ext1 were cleaned in 20% glycerol/1% KOH for 5–14 d and transferred to 50% whereas human cells begin to behave aberrantly at that level. It is glycerol/50% ethanol for photography and storage. Radiographic exami- also possible that mouse cells have a mechanism to compensate for nation was performed on a Faxitron MX-20 DC4 (Faxitron X-Ray LLC) using an inactive Ext1 allele whereas human cells cannot. In any event, an energy of 26 kV and an exposure time of 10 s. this mouse model should be useful to address these questions concerning the pathogenesis of MHE as well as to explore thera- Histology and Immunohistochemistry. Tissue specimens were fixedin4% fi fi peutic strategies for this important bone disorder. paraformaldehyde (PFA) in PBS, embedded in paraf n, decalci ed in EDTA, and sectioned at 5 mm. For Safranin O/Fast Green staining, deparaffinized, Materials and Methods rehydrated sections were stained in Weigert’sIronHematoxylin(Sigma) and 0.02% aqueous Fast Green (Sigma), followed by a rinse in 1% acetic Mice. Creation of the loxP-modified Ext1 allele (Ext1F) and Col2-CreERT acid and 0.1% aqueous Safranin O (Sigma). For immunohistochemistry, transgenic mice is described in refs. 31 and 25, respectively. From these two fixed and decalcified tissue specimens were embedded in OCT compound lines, conditional homozygous Ext1 mutant mice (Col2-CreERT;Ext1F/F; called and cryosectioned at 5 mm. After blocking with 5% goat serum, sections Ext1-SKO mice in this article) and heterozygous mutants (Col2-CreERT;Ext1F/+) were incubated overnight at 4 °C with the anti-phospho-histone H3 (pHH3) were generated. Littermates that inherited the incomplete combination of antibody (Upstate Biotechnology), followed by detection with Alexa Fluor " " the above alleles were used as controls (referred to as wild type in this 488-conjugated anti-rabbit IgG (Invitrogen) and counterstaining with 4’,6- F fi article). Throughout the article, Ext1 denotes the loxP-modi ed Ext1 allele, diamidino-2-phenylindole dihydrochloride (DAPI) (Invitrogen). Images of ΔF + – whereas Ext1 denotes the recombined null allele. Ext1 and Ext1 denote sections were taken using a TE300 Nikon fluorescence microscope. The – the wild-type and constitutive null Ext1 null alleles, respectively. The Ext1 number of pHH3-positive cells and the number of DAPI-positive nuclei were F allele was created by crossing Ext1 mice with the germline deleter Meox2- countedintwo100× 100-μm sampling windows (per section) in the car- Cre mice (32). Rosa26-lacZ (R26R) mice (26) were obtained from The Jackson tilage cap region of osteochondromas or in the growth plate. For each Laboratory. Prx1-Cre transgenic mice (33) were a gift from Cliff Tabin osteochondroma or growth plate specimens, counting was made in two (Harvard Medical School, Cambridge, MA). All experiments were done with adjacent sections, and the percentage of pHH3-positive cells to the total mice in a complete C57BL/6 background. Genotyping of mice was performed number of cells was determined for each specimen. Four osteochondroma by PCR as described in ref. 31. and four growth plate specimens were examined.

10936 | www.pnas.org/cgi/doi/10.1073/pnas.0914642107 Matsumoto et al. Downloaded by guest on September 24, 2021 X-Gal Staining. Whole-skeleton X-gal staining was performed as described in formaldehyde in PBS, decalcified, and embedded in paraffin. Sections of 10 μm ref. 34 with some modifications. Briefly, bone specimens were rapidly dis- were cut and stained with hematoxylin and eosin. Tissue fragments in the sected and prefixed in 2% glutaraldehyde for 1 h at room temperature. cartilage cap region were isolated by LCM. Special care was taken to ensure that Specimens were then incubated overnight at 4 °C in the dark in 0.1% X-gal the sample does not contain were not contaminated by cells of the calcified fi reaction buffer at pH 7.5, rinsed in PBS, and post xed in 4% PFA overnight at zone or the perichondrium. The precision of dissection was verified under the 4 °C. Specimens were placed in 1% KOH for 1–2 wk with fresh KOH every 2 d; microscope at the time of LCM. For each osteochondroma, tissue fragments the cleared samples were then stored in 70% ethanol. were pooled from four to six consecutive sections. Genomic DNA was extracted using the Fixed Tissue Genomic Isolation Kit (Promega). Semiquantitative PCR PCR. Genomic DNA was extracted from various tissues using Wizard genomic analysis was performed on these DNA samples under the following condition: 1 DNA purification Kit (Promega). For routine genotyping of animals, PCR was performed under the conditions 1 min at 94 °C, 1 min at 55 °C, and 1 min at min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C, for 60 cycles. Primers used were F ′ ′ 72 °C, for 40 cycles. Primers used were #51 (5′-GGAGTGTGGATGAGTTGAAG- #51 and #52 for the Ext1 allele, and K1 (5 -TCCCTGCTCTCTTTCTTTTC-3 )and ΔF 3′) and #52 (5′-CAACACTTTCAGCTCCAGTC-3′) for the detection of the intact #35 for the Ext1 allele. Each band was measured by densitometric analysis Δ floxed allele (Ext1F); and #51 and #35 (5′-CCAAAACTTGGATACGAGCC-3′)for using Image-J software as described in ref. 35. The ratios of Ext1 F/Ext1F were Δ the detection of the recombined floxed allele (Ext1 F). SYBR Green-based normalized, so that the result from the DNA from constitutive Ext1+/− hetero- real-time quantitative PCR analysis was carried out using the Stratagene zygous mice is 1:1. Mx3000p Sequence Detection System (Applied Biosystems) with the same set of primers for routine genotyping. The analysis of the Ext1 alleles in ACKNOWLEDGMENTS. We thank the MHE Research Foundation for sup- osteochondroma tissues isolated by laser capture microdissection (LCM) was port. This work was supported by National Institutes of Health Grants R01 performed by semiquantitative PCR. Briefly, bones were fixed in 4% para- AR055670 and P01 HD025938 (to Y.Y.).

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