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First published online 2 February 2005
doi: 10.1242/dev.01649

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Indian hedgehog synchronizes skeletal angiogenesis and perichondrial maturation with cartilage development

¹ Department of Orthopaedic Surgery, University of California, San Francisco, California, 94143-0514, USA
² Genetics Unit, Shriners Hospital, Montreal, Quebec H3G 1A6, Canada
³ Department of Plastic and Reconstructive Surgery, Stanford University, Stanford, CA 94305, USA

View larger version (109K): [in a new window]   Fig. 1. Ihh-/- chondrocytes express markers of late hypertrophy. Longitudinal sections of E16.5 (A-D) wild-type and (E-H) Ihh-/- humeri stained with (A,E) Safranin-O/Fast Green (SO) and (B-D,F-H) adjacent sections analyzed by in situ hybridization. (A) By E16.5, the primary ossification center is established in wild-type humeri as shown by the presence of the growth plate, trabecular bone (b) and periosteum (po). (B) Late hypertrophic chondrocytes and bone cells express osteopontin (Op, green) as well as (C) matrix metalloproteinase 13 (Mmp13, blue) and (D) vascular endothelial growth factor (Vegf, red). (E) Ihh-/- humeri exhibit a domain of late hypertrophic chondrocytes (lhc). No primary ossification center or distinct periosteum are observed. Ihh-/- chondrocytes express markers of late hypertrophy such as (F) Op, (G) Mmp13 and (H) Vegf, indicating that most Ihh-/- chondrocytes are not blocked in their differentiation process. B-D and F-H are high magnification views of the boxed areas in A and E, respectively. Dotted line in A-D indicates the chondro-vascular junction. Scale bars: (A,E) 200 µm, (B-D,F-H) 100 µm.

View larger version (147K): [in a new window]   Fig. 2. Ihh-/- chondrocytes undergo terminal differentiation. Longitudinal sections of (A-D) E17.5 wild-type, (E-H) E17.5 Ihh-/- and (I-K) E18.5 Ihh-/- humeri. (A,E,I,J) Sections stained with SO and adjacent sections stained with (B,F) PECAM antibody, (C,G,K) MMP9 antibody and (D,H) TRAP. (A) Morphology of the wild-type growth-plate and metaphysis. (B) PECAM immunohistochemistry shows the localization of blood vessels at the chondro-vascular junction (black, arrows), where (C) MMP9-positive (black, arrows) and (D) TRAP-positive (purple, arrows) chondro-osteoclasts accumulate. (E) In Ihh-/- humeri, islands of vascular invasion are detected within the domain of late hypertrophic chondrocytes (arrow) and co-localize with (F) PECAM-positive endothelial cells (arrow), (G) MMP9-positive (arrow) and (H) TRAP-positive cells (arrow), indicating that Ihh-/- late hypertrophic chondrocytes can induce the recruitment of endothelial cells and matrix resorbing cells. (I) By E18.5, areas of matrix degradation are seen in the center of the late hypertrophic cartilage. (J) Disintegration of the chondrocyte lacunae in these areas as well as a large number of condensed chondrocytes (arrows) suggest that Ihh-/- hypertrophic chondrocytes can undergo terminal differentiation and cell death without inducing the formation of the marrow cavity. (K) MMP9 immunostaining is detected at lower levels in the E18.5 Ihh-/- chondrocytes than it is at E17.5. Scale bars: (A,E,I) 100 µm, (B-D,F-H,K) 50 µm, (J) 20 µm.

View larger version (78K): [in a new window]   Fig. 3. Ihh-/- endothelial cells contribute to vascular invasion of cartilage but do not endure in the renal capsule environment. (A-E) Wild type;Rosa26 and (F-I) Ihh-/-;Rosa26 samples were analyzed (A-C, F-H) 4, (D,I) 10 and (E,J) 14 days after transplantation into wild-type host kidney capsules. (A) Wild type;Rosa26 cartilage elements transplanted at E14.0 are vascularized and the primary ossification center (oc) is established 4 days after transplantation, as shown by Safranin-O staining. (B) An adjacent section stained with PECAM antibody (black) shows blood vessels in the metaphysis and the periosteum. (C) Only X-gal-positive endothelial cells derived from the graft (red arrow) are detected in wild-type skeletal elements at day 4. (D) At day 10, the majority of endothelial cells are still derived from the graft (red arrow). (E) At day 14 post-transplantation, some wild-type vessels were still composed of graft-derived endothelial cells that were X-gal positive (red arrow). (F) Safranin-O staining on tissue sections shows that Ihh-/- E14.0 skeletal elements transplanted into the renal capsule for 4 days are smaller than wild-type skeletal elements but they are also vascularized and contain an ossification center (oc). (G) Blood vessels are visualized by PECAM immunostaining on an adjacent section (black). (H) At day 4, Ihh-/- blood vessels are derived entirely from graft endothelial cells. (I) At day 10, only a small proportion of X-gal-positive endothelial cells, derived from the graft, are detected in the ossification center of Ihh-/- skeletal elements (red arrow). (J) By day 14, only X-gal negative endothelial cells derived from the host are present (black arrow). Scale bars: (A,F) 200 µm, (B,G) 50 µm, (C-E,H-J) 20 µm.

View larger version (108K): [in a new window]   Fig. 4. In contrast to the in vivo environment, Ihh-/- vessels that develop ex vivo are covered by SMA-positive pericytes. (A-C) The chondro-vascular junction of E17.5 wild-type samples. (A) Collagen type IV (COL4) antibody staining highlights the invading vessels (black arrow) while (B) smooth muscle actin (SMA) antibody staining is negative. (C) An antibody against PECAM reveals endothelial cells at the junction (black arrow). (D,E) Toward the diaphysis of E17.5 elements, the marrow cavity contains vessels that are positive for both (D) PECAM and (E) SMA (red arrows). (F,G) E17.5 wild type;Ptch-lacZ samples were stained with X-gal and analyzed using SMA and PECAM antibodies. (F) Adjacent to skeletal elements, PECAM staining of endothelial cells lining blood vessels and X-gal staining in surrounding PECAM-negative cells (red arrow). (G) SMA antibody staining indicates that the X-gal-positive cells in F are pericytes (red arrow). (H-J) The vascular islands present in E17.5 Ihh-/- elements. (H) COL4 antibody identifies endothelial cells (black arrow) while (I) SMA and (J) anti-mouse control samples indicate that SMA-positive pericytes are absent. (K-M) E14 Ihh-/-;Rosa26 elements left under the kidney capsule of wild-type hosts for 10 days were stained with X-gal and analyzed by immunohistochemistry. (K) COL4 antibody staining indicates the presence of Ihh-/- endothelial cells in the ossification center (red and black arrows), and (L) SMA staining highlights a subset of these vessels as associated with SMA-positive pericytes. (M) Anti-mouse negative control samples show that the staining seen in L is specific and confirm that the vessels are derived from the graft since they are X-gal-positive (red and black arrows). In B and C, sections were counterstained with Fast-Green. lhc, late hypertrophic chondrocytes. Scale bars: (A-J) 0 µm, (K-M) 50 µm.

View larger version (121K): [in a new window]   Fig. 5. Ihh-/- cartilage elements ossify in an ex vivo environment. Histological and histochemical analyses of E14.0 (A-C) wild type; Rosa26 and (D-F) Ihh-/-;Rosa26 skeletal elements grown for 7 days in the renal capsule of adult wild-type mice. (A) Safranin-O/FG (SO) staining indicates the formation of the growth plate, periosteum (po) and trabecular bone (tb) in the metaphysis. (B) Trichrome (TC) staining shows bone matrix (blue) in the periosteum and bone trabeculae. (C) Osteoblasts (arrow) lining the bone trabeculae and osteocytes (arrowhead) embedded in the bone matrix are X-gal positive, demonstrating that osteoblasts/osteocytes are derived from the wild-type graft. (D) The Ihh-/- growth plate exhibits an abnormal organization since the zone of hypertrophic cartilage (hc) is adjacent to the articular region, however a chondro-vascular junction is seen as well as bone trabeculae and periosteum. (E) TC staining confirms the presence of bone matrix in the ossification center and the periosteum. (F) Osteoblasts (arrow) and osteocytes (arrowhead) are X-gal positive and are therefore derived from the Ihh-/- graft. B,E and C,F are higher magnifications of the boxed areas in A,D and B,E, respectively. Scale bars: (A,D) 200 µm; (B,E) 100 µm; (C,F) 20 µm.

View larger version (110K): [in a new window]   Fig. 6. Early osteoblast markers are expressed in the Ihh-/- perichondrium. (A,E) Trichrome (TC) staining and (B-D, F-H) in situ hybridization analyses on longitudinal sections of E18.5 (A-D) wild-type and (E-H) Ihh-/- humeri. (A) In wild-type samples, the bone matrix in the periosteum (po) and trabecular bone (tb) stains blue with TC. (B-D) On adjacent sections, bone cells in the periosteum and the bone trabeculae (tb) express (B) collagen type 1 (Col1, pink), (C) neuropilin 2 (Nrp2, yellow) and (D) osteocalcin (Oc, green). Dotted line in (A-D) indicates the chondro-vascular junction. (E) Weak aniline blue staining is observed in the perichondrium of Ihh-/- humeri (delimited by a red dotted line), mainly where the perichondrium is juxtaposed to the domain of late hypertrophic chondrocytes (lhc). (F) Col1 and (G) Nrp2 are expressed in the Ihh-/- perichondrium, but (H) Oc expression is not detected, indicating that osteoblast precursors are present but do not differentiate into mature osteoblasts. Scale bar: (A-H) 100 µm.

View larger version (65K): [in a new window]   Fig. 7. The hedgehog pathway is not ectopically activated ex vivo. Morphological and histological analyses of (A-E) wild type;PtchlacZ and (F-J) Ihh-/-;PtchlacZ E14.0 cartilage elements that were transplanted for (A-D,F-I) 2 or (E-J) 14 days into the renal capsule of wild-type adult mice, in the (C,D,H,I) presence or (A,B,E,F,G,J) absence of recombinant SHH-N protein. (A) Whole-mount X-gal staining shows Ptch expression throughout a wild-type cartilage element. (B) A section of the same sample shows Ptch expression (blue staining) in the perichondrium (pc) and the cartilage (c). (C) When SHH-N beads are implanted adjacent to the cartilage element (blue beads), X-gal staining is increased around the beads. (D) Tissue sections confirm the up-regulation of Ptch in cells adjacent to the bead (arrow). (E) At 14 days after transplantation, wild type;PtchlacZ cartilage elements have become ossified. Some osteoblasts in the periosteum (arrowhead) are X-gal-positive, though osteoblasts in the endosteum (red arrow) and osteocytes (black arrow) are X-gal-negative, indicating that Ptch is not expressed in mature osteoblasts. (F) Ihh-/- cartilage elements exhibit low background levels of X-gal staining 4 days after transplantation into the renal capsule. (G) X-gal staining was not detected in the perichondrium or the cartilage in tissue sections, indicating that no HH signal is provided in the graft tissue or in the renal capsule environment. (H) In the presence of SHH-N beads, Ptch is up-regulated as shown by strong X-gal staining around the beads. (I) Cells in the perichondrium and in the mesenchyme surrounding the beads are X-gal-positive, demonstrating that the Ihh-/- tissue is responsive to a HH signal. (J) The Ihh-/- tissue remains X-gal-negative even after 14 days ex vivo, confirming the absence of HH signal in the renal capsule environment. In the diaphysis of Ihh-/- skeletal elements, all osteoblasts in the periosteum (arrowhead) and endosteum (red arrow) as well as osteocytes (black arrow) are X-gal-negative, suggesting that osteoblast differentiation can occur in the absence of Ptch up-regulation. Scale bars: (A,C,F,H) 1 mm; (B,D,E,G,I,J) 50 µm.

View larger version (95K): [in a new window]   Fig. 8. The perichondrial vasculature is defective in Ihh-/- endochondral skeleton. Transverse sections of (A-E) wild-type and (F-J) Ihh-/- autopods at (A,B,F,G) E12.5 and (C-E,H-J) E14.0, analyzed by (A,C,F,H) SO staining, (B,D,G,I) in situ hybridization and (E,J) PECAM immunostaining. (A) By E12.5, flattened perichondrial cells are well organized around the wild-type cartilage condensations, and (B) express the Ihh receptor patched (Ptch, red); chondrocytes express Ptch and Ihh (yellow). (C) By E14.0, the wild-type perichondrium is composed of several cell layers where (D) Col1 is expressed highly (blue) surrounding the Col2-expressing cartilage (pink). Col2 starts to be down-regulated in hypertrophic chondrocytes (arrow). (E) The perichondrium contains PECAM-positive endothelial cells (brown, arrows) adjacent to the hypertrophic cartilage. (F) At E12.5, fewer perichondrial cells are seen around the Ihh-/- cartilage condensation compared to wild type, and they do not appear to condense properly to form a perichondrium. (G) Ihh and its target gene Ptch are not expressed, representing the absence of HH signaling in the Ihh-/- developing long bones. Hoechst dye staining of nuclei (blue) shows the morphology of the cartilage and the perichondrium. (H) By E14.0, only a thin layer of perichondrial cells surrounds the Ihh-/- cartilage. (I) A broad, more diffuse domain of Col1 expression surrounds the Col2 expressing cartilage of Ihh-/- elements, suggesting that the mutant perichondrium is defective at a molecular level. (J) PECAM-positive endothelial cells are excluded from the Ihh-/- perichondrium. Scale bars: (A-D,F-I) 100 µm; (E,J) 200 µm.

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