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First published online 18 February 2004
doi: 10.1242/dev.01006

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Ihh signaling is directly required for the osteoblast lineage in the endochondral skeleton

Fanxin Long^1,*,, Ung-il Chung^2,*,, Shinsuke Ohba³, Jill McMahon¹, Henry M. Kronenberg² and Andrew P. McMahon^1,

¹ Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
² Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
³ Department of Bone and Cartilage Regenerative Medicine, University of Tokyo School of Medicine, Tokyo, Japan

View larger version (83K): [in a new window]   Fig. 1. Morphological analyses of the bone collar. (A-D) Whole-mount skeletal staining of (A-C) the radius (R) and ulna (U) and (D) the tibia at E18.5. Alizarin Red stains the bone red and Alcian Blue stains the cartilage blue. (C) The bone collar in Cre10; Smon/c is separated from the cartilage (orange arrowhead) and contains cartilage in certain areas (green arrowhead). (D) No bone collar (red arrowhead) is formed in the long bones of Ihhn/n embryos. The mineralized hypertrophic cartilage (H) is stained dark red. (E-I) Mineralization revealed by von Kossa staining (black) on sections of E18.5 tibia counterstained with Methyl Green. Genotypes for E-G correspond to A-C, respectively. (G) The bone collar in Cre10; Smon/c is separated from the marrow cavity (M) by several layers of cells (orange double arrowhead). (H,I) These cells assume the morphology of chondrocytes (I, a higher magnification of the boxed region in H. B, bone; C, chondrocytes; M, marrow). The asterisk in H denotes a region where the bone collar is missing. (J) In situ hybridization using 35S-labeled riboprobes against Col1(II) on a section adjacent to that in H. High levels of expression (signal in red) are detected in cells separating the bone collar and the marrow cavity in Cre10; Smon/c embryos (between arrows). (K,L,M) Analyses of Cre activity of the Cre10 line using Rosa26 reporter (R26R) mice. Evidence of Cre activity (strong blue staining) is detected in chondrocytes (C) as well as in the perichondrium (PC) and the periosteum (PO), whereas muscles (M) and tendons (T) are negative. A higher magnification (L) of the boxed region in K shows strong Cre activity in osteoblasts of the bone collar (red arrowheads). Strong Cre activity is also evident in osteoblasts of the primary spongiosa (red arrowheads in M).

View larger version (122K): [in a new window]   Fig. 2. In situ hybridization of 35S labeled riboprobes to limb elements following Smoothened removal. (A-J) E14.5 tibial elements of indicated genotypes hybridized with probes as indicated. Signals are shown in red on sections counterstained with Toluidine Blue. The orange arrows denote the perichondrium not expressing the various genes in Cre10; Smon/c embryos.

View larger version (148K): [in a new window]   Fig. 3. Analysis of skeletal elements in chimeric embryos. Contribution of Smon/n (A-H) or Smon/+ (J) cells to the bone collar of long bones (A-F,J) and to the mandible (G,H) in chimeric embryos at E17.5. Smon/n and Smon/+ cells are stained blue as a result of the expression of ß-galactosidase and the sections are counterstained with Nuclear Fast Red (A,B,G,H), H&E (C,J), or Methyl Green (D). Mineralized tissues are stained black by the von Kossa method (A,B,G,H,J). (A,B) The bone collar forms on one side of the tibia (A, arrowheads) or the fibula (B, arrowheads), but fails to develop from the perichondrium on the opposite side which is composed of mutant cells. (C) The Smon/n perichondrial cells differentiate into chondrocytes (arrows). (D) Safranin-O stains red the cartilaginous matrix deposited by chondrocytes differentiated from the Smon/n perichondrial cells. (E,F) Dark-field images of in situ hybridization using 35S-labeled riboprobes for Col1(II) (E) and Col 1(X) (F) on sections similar to that in D. (G,H) Smon/n cells contribute to the osteoblast population of the mandible. Asterisk in G denotes Meckel's cartilage. The boxed region in G is shown at a higher magnification in H. (J) In chimeras composed of Smon/+ and wild-type cells, the Smon/+ cells contribute to the bone collar (black arrowheads).

View larger version (151K): [in a new window]   Fig. 4. Lack of primary spongiosa in Cre10; Smon/c embryos. (A-D) H&E staining of longitudinal sections through the tibia at P3. The boxed regions in A and B are shown at a higher magnification in C and D. The straight lines in C and D define a region of the same length that in the wild type (C) but not in the mutant (D) is populated by cells with morphology that is distinct from that of the marrow cells. (E-H) In situ hybridization using 35S labeled riboprobes for osteocalcin (E,F) and TRAP (G,H) on longitudinal sections of the tibia at P3. Signals are shown in red and the sections are counterstained with Toluidine Blue.

View larger version (141K): [in a new window]   Fig. 5. Analysis of Hh response and Ihh expression in skeletal elements. In situ hybridization using 35S-labeled riboprobes for Ihh (A,B) and Ptc1 (CE) on longitudinal sections of the tibia at P3. Signals are shown in red and the sections are counterstained with Toluidine Blue. The boxed region in C is shown at a higher magnification in E. The straight lines in E demarcate the region populated by cells with morphology that is distinct from that of the marrow cells, similar to that defined in C and D of Fig. 4.

View larger version (66K): [in a new window]   Fig. 6. Analysis of the primary spongiosa in chimeric embryos. Smon/n cells fail to contribute to the primary spongiosa in chimeric embryos (A,B) whereas Smon/+ cells contribute extensively (C). Longitudinal sections of the tibia at E17.5 are stained with H&E. Smon/n or Smon/+ cells are stained blue as a result of the expression of ß-galactosidase. The boxed region in A is shown at a higher magnification in B. Asterisks denote the blood vessels. Arrows in B point to occasional Smon/n cells in the marrow cavity.

View larger version (170K): [in a new window]   Fig. 7. Analysis of bone formation in the perichondrium following activation of Ihh signaling. (A-H) In situ hybridization using 35S-labeled riboprobes on longitudinal sections of the tibia at E16.5; probes and genotypes as indicated. Signals are shown in red and the sections are counterstained with Toluidine Blue. H, hypertrophic chondrocytes; M, marrow cavity. Asterisks denote the perichondrium in which Ptc1 or Cbfa1 are up-regulated.

View larger version (90K): [in a new window]   Fig. 8. Interaction of Hh and BMP pathways during osteoblast differentiation in vitro. Total RNA extracted from MLB13MYC clone 17 cells treated with various reagents were subjected to northern analyses (see Materials and methods). GAPDH is used as a control for loading and transfer efficiency.