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. 2010 Feb 2;107(5):2054-9.
doi: 10.1073/pnas.0910875107. Epub 2009 Dec 22.

A mouse model of osteochondromagenesis from clonal inactivation of Ext1 in chondrocytes

Kevin B Jones  1 , Virginia Piombo, Charles Searby, Gail Kurriger, Baoli Yang, Florian Grabellus, Peter J Roughley, Jose A Morcuende, Joseph A Buckwalter, Mario R Capecchi, Andrea Vortkamp, Val C Sheffield
Affiliations

A mouse model of osteochondromagenesis from clonal inactivation of Ext1 in chondrocytes

Kevin B Jones et al. Proc Natl Acad Sci U S A. .

Abstract

We report a mouse model of multiple osteochondromas (MO), an autosomal dominant disease in humans, also known as multiple hereditary exostoses (MHE or HME) and characterized by the formation of cartilage-capped osseous growths projecting from the metaphyses of endochondral bones. The pathogenesis of these osteochondromas has remained unclear. Mice heterozygous for Ext1 or Ext2, modeling the human genotypes that cause MO, occasionally develop solitary osteochondroma-like structures on ribs [Lin et al. (2000) Dev Biol 224(2):299-311; Stickens et al. (2005) Development 132(22):5055-5068]. Rather than model the germ-line genotype, we modeled the chimeric tissue genotype of somatic loss of heterozygosity (LOH), by conditionally inactivating Ext1 via head-to-head loxP sites and temporally controlled Cre-recombinase in chondrocytes. These mice faithfully recapitulate the human phenotype of multiple metaphyseal osteochondromas. We also confirm homozygous disruption of Ext1 in osteochondroma chondrocytes and their origin in proliferating physeal chondrocytes. These results explain prior modeling failures with the necessity for somatic LOH in a developmentally regulated cell type.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Generation and characterization of head-to-head floxed Ext1. (A) Homology arms procured by long-range PCR (small arrows: PCR primers) from genomic Ext1 DNA (white boxes, exons; black lines, introns) were subcloned into pOSDUPDEL to either side of the neomycin resistance cassette (neo) flanked by forward orientation loxP sites (triangles). An inverted loxP site was introduced upstream of exon 2. _targeting was tested with long-range PCR. (B) Cre-mediated recombinations (gray arrows) produce a distribution of products (black boxes: inverted sequence) from any given state, including entire construct inversion, exon 2 inversion, or neo excision. After neo excision (e2fl or e2flinv), only exon 2 inversion and reversion are possible. Each of the recombination states is detectable by the primer pairs listed below it (small arrows). (C) PCR using template DNA isolated from Ext1e2neofl/+ MEFs after treatment with TATCre demonstrates presence of all recombination states. (D) Ext1e2neofl/e2neofl MEFs after TATCre demonstrated the normal diploid murine karyotype of 40 chromosomes counterstained with DAPI including 2 stained with FISH chromosome 15 paint (magenta).
Fig. 2.
Fig. 2.
Exon 2 inversion disrupts the function of Ext1. Immunohistochemistry of E7.0 frontal sections of wild-type (A) and Ext1e2flinv/e2flinv (B) embryos with 10E4 antibody demonstrated failing gastrulation and reduced HS in the embryonic tissue (arrows) of Ext1e2flinv/e2flinv, whereas placental tissues showed intensive staining for HS (ec, exocoelomic cavity; pc, proamniotic cavity; e, ectoderm; ps, primitive streak; bs, basement membrane; en, endoderm). (Scale bar, 50 μm.)
Fig. 3.
Fig. 3.
Postnatal inactivation of Ext1 in chondrocytes generates osteochondromas. (A) Three-dimensional microCT rendering of a 10-week-old Col2-rtTA-Cre;Ext1e2neofl/e2neofl mouse after doxycyline treatment shows osteochondromas on the distal femur and proximal tibia (arrows). Coronal plane (B) and axial plane (C) reconstructions of the distal femur confirm cortical and medullary continuity of the osteochondromas, matching radiographic definitions for human diagnosis. Safranin-O stained sections from the knees of 4- (D), 6- (E), and 10-week-old (F) Col2-rtTA-Cre;Ext1e2neofl/e2neofl mice show early, middle, and fully developed osteochondromas, respectively, and both pedunculated (F, black arrow), and sessile (F, white arrow) morphologies.
Fig. 4.
Fig. 4.
The proliferating chondrocyte is the cell of origin for an osteochondroma. (A) β-Gal staining of a P16 proximal tibial physis from Col2-rtTA-Cre;Rosa26LacZ/+ after P8–12 doxycyline by drinking water shows abundant staining of proliferating chondrocytes (C) with minimal staining of the inner layer of cells in the perichondrium/periosteum (P). (B) Osx-CreERT;Rosa26LacZ/+ mice after P8 injection of tamoxifen have more robust staining of the inner layer of perichondrium/periosteum (P), with additional staining of hypertrophic chondrocytes (H), but not proliferating chondrocytes. (C) Rosa26LacZ/+ mice without Cre-recombinase demonstrate only mild background staining of primary spongiosal osteoblasts. Three-dimensional knee renderings of microCTs from 9-week-old Col2-rtTA-Cre;Ext1e2neofl/e2neofl (D), Osx-CreERT;Ext1e2neofl/e2neofl (E), and Ext1e2neofl/e2neofl (F) mice demonstrate the presence of osteochondromas (arrows) only on the Col2-rtTA-Cre mutants. Safranin-O staining from the same groups confirms the presence (G, black arrows) and absence (H and I) of osteochondromas, but also the presence of a small epiphyseal enchondroma (H, white arrow) in the Osx-CreERT mutant, likely from its hypertrophic chondrocyte Cre activity. (Scale bars: A–C, 100 μm; D–I, 500 μm; background artifact has been removed from panel 4G).
Fig. 5.
Fig. 5.
Osteochondroma chondrocytes demonstrate clonal inversion of exon 2 of Ext1. (A and B) Immunohistochemistry with 10E4 antibody in parallel sections showed that HS negativity correlated with homozygosity for the inverted allele (red-encircled clones). HS-positive chondrocytes (D, enlarged from C) demonstrated the Ext1e2fl/e2fl (yellow-encircled clones) genotype. (E) The distribution of 55 LCM-identified clone genotypes from osteochondroma chondrocytes demonstrates a strong majority of Ext1e2flinv/e2flinv genotypes at all ages. (Scale bars, 100 μm.)
Fig. 6.
Fig. 6.
Osteochondromagenesis. (A) An early proliferative chondrocyte with homozygous disruption of Ext1 has no HS on its cell surface or in the immediate extracellular matrix (black clone of cells). (B) This loss of HS delays the clone’s differentiation and gives it a proliferative advantage. The immediately adjacent perichondrium fails to differentiate into osteoblasts and form a bone collar (white asterisk) due to blocked osteogenic signals from the physis, physical displacement away from the osteogenic signals from the physis, or receipt of alternate signals from the less-differentiated chondrocytes of the early osteochondroma. (C) Once the clone has progressed past the hypertrophic zone, the physis-flanking perichondrium receives normal osteogenic signals (black asterisks) again and reforms the bone collar in the wake of the osteochondroma. The osteochondroma may or may not have brought along cells of normal genotype (white cells, C and D) from the physis. (D) The fully developed osteochondroma, once completely independent from direct physeal signaling, begins to organize itself loosely into the proliferative (P) and hypertrophic (H) zones of a growth plate, as well as form its own primary spongiosa and bone collar, each in continuity with the host bone.
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Comment in

  • EXTra hit for mouse osteochondroma.
    Bovée JV. Bovée JV. Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):1813-4. doi: 10.1073/pnas.0914431107. Proc Natl Acad Sci U S A. 2010. PMID: 20133829 Free PMC article. No abstract available.

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