Volume 8, Issue 5, Pages 727-738 (May 2005) Canonical Wnt/β-Catenin Signaling Prevents Osteoblasts from Differentiating into Chondrocytes  Theo P. Hill, Daniela Später, Makoto M. Taketo, Walter Birchmeier, Christine Hartmann  Developmental Cell  Volume 8, Issue 5, Pages 727-738 (May 2005) DOI: 10.1016/j.devcel.2005.02.013 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Endogenous β-Catenin Expression in Skeletal Tissue and Prx1-Cre Activity (A–A″) Immunohistochemical staining showing higher levels of β-catenin protein in cells of the (A) periarticular region of the joint, in the (A′ and star in A″) nucleus of flattened proliferating and prehypertrophic chondrocytes, and in the (A″) periosteum at E13.5. (B and C) Alkaline phosphatase staining of ZAP-reporter/Prx1-Cre embryos at (B) E9.5 and (C) E11. (D) Coronal section through the head at the level of the white line shown in (C). (E and E′) In situ hybridization showing β-catenin expression in a (E) wild-type hindlimb and in a (E′) β-catΔPrx/− hindlimb at E11.5. Arrows indicate the expression in cells that are probably migrating myoblasts. Developmental Cell 2005 8, 727-738DOI: (10.1016/j.devcel.2005.02.013) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Skeletal Alterations in β-Catenin Mutant Animals at Late Stages of Development (A–C) Skeletal preparations of wild-type (WT) and β-catΔPrx/− (ΔPrx/−) mutant P0 embryos showing the whole skeleton, the hindlimbs, and the head skeleton. (D–G) Wild-type and mutant femurs at PO. (D) Hematoxylin/eosin staining. (E) Van Kossa staining of bone collar in the wild-type (arrow), which is absent (star) in the mutant. (F) TRAP staining, demonstrating that multinucleated osteoclasts (inlet) are present in the mutant. High-magnification (20×) hematoxylin/eosin-stained sections, showing that eosin-positive red blood cells and hematopoietic cells are present (E). Developmental Cell 2005 8, 727-738DOI: (10.1016/j.devcel.2005.02.013) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Absence of Mature Osteoblast Markers in β-Catenin Mutant Limbs and Mineralization Defects of Femora Cultures (A–I) Nonradioactive section in situ hybridizations on wild-type and β-catΔPrx/− (ΔPrx/−) mutant femora at (A–C) E14.5, (D–G) E16.5, and (H and I) E18.5, showing expression of (A) Ihh, (B and E) Runx2, (C and F) Osx, (D) Ptc-1, (G) Tcf-1, (H) Col1a1, and (I) Osc. Note that the folds within the skeletal elements are due to sectioning and do not reflect increased expression. (J) Alizarin red-stained mineralization assay of cultures from β-catenin heterozygous and mutant ΔPrx/− femora at days (d) 15 and 21 of differentiation, without (w/o) addition of exogenous factors and in the presence of exogenous recombinant Shh and BMP2 protein; this panel shows decreased mineralization potential of mutant cultures. (K) Relative expression of Osteocalcin (Osc), Alkaline phosphatase (ALP), and bone sialoprotein (Bsp) in heterozygous (blue) and mutant cultures (red) at day 6, without the addition of factors (solid bars) and with the addition of Shh and BMP2 (stippled bars). Error bars represent the standard deviation of the mean of the results (n = 3). Developmental Cell 2005 8, 727-738DOI: (10.1016/j.devcel.2005.02.013) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Differentiation of Perichondrial/Periosteal Cells into Chondrocytes in β-Catenin Mutants (A–C) Sections through wild-type and β-catΔPrx/− (ΔPrx/−) mutant femora at E18.5 stained with van Kossa/alcian blue at low magnification. High magnification showing alcian blue-positive cells within the mutant perichondrium/periosteum (arrow in [A′]), which express Col2a1 (arrow in [B]) and Sox9 (arrow in [C]). (D–F) (D) Ihh and (E) Col10a1 are only expressed in cells along the wedge-shaped mutant perichondrium, which can be also seen in the (F) hematoxylin/eosin-stained section. Col2a1, Sox9, Ihh, and Col10a1 expression are visualized by in situ hybridization. Developmental Cell 2005 8, 727-738DOI: (10.1016/j.devcel.2005.02.013) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Loss of β-Catenin in Skull Mesenchyme and Calvarial Osteoblast Progenitors Leads to Ectopic Cartilage Formation (A) Coronal sections through the skull of wild-type and β-catΔPrx/− (ΔPrx/−) mutants at P0 stained with van Kossa alcian blue (blue), showing the presence of mineralized bone (arrow) in the wild-type, which is absent in the mutant and replaced by ectopic cartilage. (B–D) In situ hybridizations on coronal sections showing a dorsal expansion of chondrogenic markers (B) Sox 9 and (C) Col2 in the mutants (see arrows) at E13.5 and (D) loss of Osx expression at E16.5. (E) Whole-mount in situ hybridizations on E16.5 wild-type and β-catΔPrx/− mutant heads, showing lack of Osc expression (arrow) in the mutant skull. (F) Primary osteoblast cultures from β-catenin fl/fl E16.5 calvariae treated with AdGfp (left panel) and AdCre (right panel) showing different morphology at (Fa) day 15 and formation of alcian blue-positive nodules at (Fb) day 21. In situ hybridization for (Fc) Col2a1, (Fd) Sox9, and (Fe) Col1a1. (G) Relative expression levels of chondrogenic markers Sox9 and Col2a1 and of the marker Col1a1 at days 5, 10, and 15 of culture, analyzed by real-time PCR. Error bars represent the standard deviation of the mean of the results (n = 2). Developmental Cell 2005 8, 727-738DOI: (10.1016/j.devcel.2005.02.013) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Alterations of Prechondrogenic Marker Expression in β-Catenin Gain- and Loss-of-Function Mutant Limb Buds (A–J) Transverse sections through the forelimb of (A–D) wild-type, (E–G) β-catenin gain-of-function (Δex3Prx/+) mutants, and (H–J) β-catenin loss-of-function (ΔPrx/−) mutants at E11.0. (A) Hematoxylin/eosin staining visualizing the condensed mesenchyme in the core of a wild-type limb. (B) In situ hybridization showing Sox9 expression in the condensed core. (C) Immunohistochemical staining showing higher levels of β-catenin protein in the mesenchyme underneath the ectoderm, and lower levels in the central core. (D) In situ hybridization showing Runx2 expression in the scapula region. (E) No condensation is visible in β-catΔex3Prx/+ mutant limb buds. (F) Sox9 expression is downregulated in β-catΔex3Prx/+ mutant limbs. (G) Ectopic stabilization of β-catenin protein in β-catΔex3Prx/+ limbs. Mosaic activity of the Prx1-Cre line is reflected by the presence of a few clusters of cells, which do not show ectopic stabilization of β-catenin. (H) No condensation is visible in β-catΔPrx/− mutants, and (I) Sox9 expression is expanded in the distal half of the limb bud. (J) Expanded expression of Runx2 in β-catΔPrx/− mutant limb bud. Developmental Cell 2005 8, 727-738DOI: (10.1016/j.devcel.2005.02.013) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Alteration of β-Catenin Levels in Micromass Cultures Affects Sox9 and Runx2 Expression Levels (A) Western blot showing reduction of β-catenin levels in AdCre-treated cultures from 24 hr onward. (B) Time course of real-time PCR analysis showing upregulation of Sox9 starting at 24 hr in AdCre-treated β-catenin fl/fl cultures (n = 2). (C) Alcian blue-stained micromass cultures at days 4 and 6 from β-catenin fl/fl limbs infected with AdenoGfp or AdenoCre virus with and without (w/o) addition of rhBmp4, showing accelerated cartilage nodule formation upon deletion of β-catenin. (D) Western blot showing the appearance of the N-terminal-deleted β-catenin in ex3fl/ex3fl AdCre-treated cultures at 10 hr. (E) Real-time PCR analysis showing downregulation of Sox9 from 10 hr onward in AdCre-treated β-catenin ex3fl/ex3fl cultures (n = 3). (F) Real-time PCR analysis showing relative expression levels of Runx2 at 14 hr going up in AdCre-treated β-catenin fl/fl cultures and going down in AdCre-treated β-catenin ex3fl/ex3fl cultures. Error bars represent the standard deviation of the mean of the results (n = 3). (G) Semiquantitative RT-PCR analysis of Lef-1 expression, showing an upregulation of Lef-1 in AdCre-treated β-catenin ex3fl/ex3fl at 14 hr of culturing. Developmental Cell 2005 8, 727-738DOI: (10.1016/j.devcel.2005.02.013) Copyright © 2005 Elsevier Inc. Terms and Conditions