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First published online August 25, 2006
doi: 10.1242/10.1242/dev.02546

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Wnt/ß-catenin signaling interacts differentially with Ihh signaling in controlling endochondral bone and synovial joint formation

¹ Genetic Disease Research Branch, National Human Genome Research Institute, Bethesda, MD 20892, USA.
² Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA.

View larger version (20K): [in a new window]   Fig. 1. Generation of a conditional allele of mouse Ptch1. Schematic diagram showing the mouse patched 1 (Ptch1) genomic locus, the targeting vector and the mutant alleles. The top line shows a partial restriction map of the mouse Ptch1 genomic locus on chromosome 13. The mouse Ptch1 genomic locus consists of 23 exons (E1-E23) and only the first two exons are shown for simplicity. The translation start ATG is predicted to reside in the first exon (E1). The regions between the dotted lines represent the 5' and 3' regions of homology used in gene targeting, respectively, and `X' indicates events of homologous recombination. Germline-transmitting chimeric males carrying the targeted Ptch1 locus were mated with FLPe (Rodriguez et al., 2000) mice to remove the PGKneo selection cassette, which is flanked by two FRT sites. The resulting allele is a conditional allele of Ptch1, denoted as Ptch1c. Ptch1c/+ animals were mated with a Cre deleter strain [e.g. ß-actin::Cre (Meyers et al., 1998)] to remove sequences between the two loxP sites and to generate a null allele, designated as Ptch1- or Ptch1loxP in this study. Matings were set up between Ptch1loxP/+ animals, and homozygous Ptch1loxP/loxP embryos display characteristic phenotypes that are indistinguishable from those of the published null alleles of Ptch1 (Goodrich et al., 1997). When the Ptch1 conditional allele is converted into the null allele, ß-galactosidase is brought under the control of the Ptch1 regulatory elements. It should be noted that a small percentage of homozygous Ptch1c/c animals display embryonic/neonatal lethality, suggesting that the inclusion of lacZ may have affected the expression from the Ptch1 locus.

View larger version (86K): [in a new window]   Fig. 2. Inactivation of Ptch1 during endochondral skeletogenesis resulted in multiple defects. (A-H) Sections of developing long bones. (A,B) Safranin O staining of sections of humerus from 18.5 dpc embryos at low magnification. Proliferative chondrocytes were bright red, whereas hypertrophic chondrocytes were light red. Hypertrophic chondrocytes were not detected in the Ptch1c/-; Col2a1-Cre mutant. (C,D) Joint regions between radius and carpel bones at 14.5 dpc are shown at higher magnification. Robust upregulation of Pthrp expression in the articular cartilage was observed in the mutant (arrow, D). (E,F) von Kossa staining of the distal humerus at 18.5 dpc. Ectopic ossification was observed in the joint of the mutant (arrow, F). (G,H) Safranin O staining of the radius/ulna/carpel junction at 18.5 dpc. Radius and ulna were fused with carpel bones in the mutant (arrows). (I-N) Skeletal preparations of 18.5 dpc wild-type (I-K) and Ptch1c/-; Col2a1-Cre (L-N) mouse embryos. (I,L) Whole embryos; (J,M) forelimbs; (K,N) hindlimbs. Inactivation of Ptch1 resulted in extensive ossification. Ectopic ossification at the joint is indicated by arrows (M,N). Skull formation was defective in the mutant (arrowhead, L). S, scapula; H, humerus; R, radius; U, ulna; Fe, femur; Fi, fibula; Ti, tibia.

View larger version (93K): [in a new window]   Fig. 3. Analysis of Ptch1 and ß-catenin mutant skeletons. (A) Skeletal preparations of 16.5 dpc mouse embryos. A forelimb of each embryo is shown in the lower panel. The posterior skull in the Ptch1c/-; Col2a1-Cre mutant did not form (arrow). Skull formation in the Ptch1c/-; Catnbc/-; Col2a1-Cre double mutant was rescued (arrow). Mineralization (arrows, lower panel) in the long bones was more severely reduced in the Ptch1c/-; Catnbc/-; Col2a1-Cre double mutant than in either of the single mutants. S, scapular; H, humerus; R, radius; U, ulna. (B) ß-catenin is not required for Ihh signaling. Consecutive sections of the developing humerus at 14.5 dpc were examined by in situ hybridization with the indicated 35S-labelled riboprobes. In both Ptch1c/-; Col2a1-Cre and Ptch1c/-; Catnbc/-; Col2a1-Cre mutant embryos, expression of the Hh signaling targets Hip1 and Gli1, and of Pthrp, was similarly upregulated in cartilage, perichondrium and joints (arrows). The perichondrium was slightly thinner in the Ptch1c/-; Catnbc/-; Col2a1-Cre mutant than in the Ptch1c/-; Col2a1-Cre mutant.

View larger version (81K): [in a new window]   Fig. 4. Analysis of osteoblast differentiation in ß-catenin and Ptch1 mutant embryos. (A,B) Consecutive sections of the developing humerus at 14.5 dpc (A) and 16.5 dpc (B) were examined by von Kossa staining and in situ hybridization with the indicated 35S-labelled riboprobes. (A) At 14.5 dpc, ColX expression was missing, and expression of the early osteoblast markers Runx2 and Osx was activated in both Ptch1c/-; Col2a1-Cre and Ptch1c/-; Catnbc/-; Col2a1-Cre mutants. Ossification and expression of the late osteoblast markers Opn and Mmp13 were only detected in the wild-type embryo. (B) At 16.5 dpc, ColX expression was still missing in the Ptch1c/-; Col2a1-Cre and Ptch1c/-; Catnbc/-; Col2a1-Cre mutants. Osx expression was still stronger in the Ptch1c/-; Catnbc/-; Col2a1-Cre mutant than in the Catnbc/-; Col2a1-Cre mutant. Ossification and expression of Opn and Mmp13 were enhanced in the Ptch1c/-; Col2a1-Cre mutant, but diminished in Catnbc/-; Col2a1-Cre and Ptch1c/-; Catnbc/-; Col2a1-Cre mutants.

View larger version (40K): [in a new window]   Fig. 5. Enhanced delay of chondrocyte hypertrophy in ß-catenin and Ptch1 double mutant embryos. Consecutive sections of the developing tibia at 14.5 dpc were examined by Safranin O staining and in situ hybridization with the indicated 35S-labelled riboprobes. Hypertrophic chondrocytes are enlarged and stained light red by Safranin O. The expression domains of Ihh and ColX were smaller in both Ptch1c/-; Col2a1-Cre and Catnbc/-; Col2a1-Cre mutant embryos, and were missing in the Ptch1c/-; Catnbc/-; Col2a1-Cre double mutant (arrows). Pthrp expression was similarly upregulated in Ptch1c/-; Col2a1-Cre and Ptch1c/-; Catnbc/-; Col2a1-Cre mutants (arrows).

View larger version (53K): [in a new window]   Fig. 6. Analysis of chondrocyte proliferation in ß-catenin and Ptch1 mutant embryos. (A) BrdU-labelled cells were detected by immunohistochemistry on sections of the developing tibia at 14.5 dpc. Zone I (resting zone) contains slow proliferating chondrocytes and Zone II (proliferative zone) contains fast proliferating chondrocytes. For each genotype, BrdU-labelled and total chondrocyte numbers in the boxed regions were counted from three different samples to find the mean. (B) Comparison of BrdU-labelled chondrocytes in Zone I and II in different mutants. Three samples were counted, and the mean±s.d. and statistically significant P-values are shown.

View larger version (97K): [in a new window]   Fig. 7. Expression of cell cycle regulators and apoptosis in ß-catenin and Ptch1 mutant embryos. Results of immunohistochemistry with the indicated antibodies on limb sections of the indicated genotypes is shown. (A) Expression of the indicated cell cycle regulators in the distal humerus at 14.5 dpc. Boxed areas representing negative (left) and positive (right) staining regions in the wild-type sample and corresponding regions in every mutant are shown at higher magnification as insets in the lower left and right corners, respectively. (B) Expression of p21 and detection of cell death by fluorescent TUNEL assays on tibia sections at 18.5 dpc. Apoptotic cells are stained green; nuclei are stained blue by DAPI. p21 expression and apoptosis were greatly increased in the Catnbc/-; Col2a1-Cre mutant.

View larger version (107K): [in a new window]   Fig. 8. Analysis of joint formation in ß-catenin and Ptch1 mutant embryos. (A) Expression of Hip1 in the developing joint (arrows) at 14.5 dpc, as shown by in situ hybridization with 35S-labelled riboprobes. (B) Safranin O staining. Complete fusion of the humerus-ulna joint was observed only in the Ptch1c/-; Catnbc/-; Col2a1-Cre mutant (arrow). (C) Consecutive sections of the developing humerus-ulna region at 14.5 dpc were examined by in situ hybridization with the indicated 35S-labelled riboprobes. Upregulation of Bmp2, Bmp4 and Bmp7 (arrows), but not Gdf5, was observed in both Ptch1c/-; Col2a1-Cre and Ptch1c/-; Catnbc/-; Col2a1-Cre mutants. (D) Safranin O staining of sections of the forelimbs that were isolated from 12.5 dpc embryos and cultured in vitro for 4 days. Boxed regions in the humerus-scapula joint are shown at higher magification in the insets. Joint cells are indicated (arrows). The fused joint in the Ptch1c/-; Col2a1-Cre limb was rescued by noggin treatment. H, humerus; S, scapula. (E) Micromass culture with cells from Catnbc/c embryonic limb bud mesenchyme at 12.5 dpc. Cartilage nodules were stained blue with Alcian Blue. ß-catenin was deleted by Cre-Adenovirus infection. Bmp2 and noggin were added to the culture on day 2 of the 5-day culture period.

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