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TGFß2 mediates the effects of Hedgehog on hypertrophic differentiation and PTHrP expression

¹ Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0576, USA
² Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0524, USA

View larger version (98K): [in a new window]   Fig. 1. Effects of Shh treatment on embryonic mouse metatarsal bone rudiments. Mouse embryonic metatarsal explants were either untreated (A,C,E,G) or treated with 2 µg/ml recombinant Shh (B,D,F,H) for 5 days. (A,B) Morphology. In control cultures, a clear area of hypertrophic cartilage was observed in the center of the bone rudiments and matrix mineralization was observed in the center of the hypertrophic cartilage (dark area; A). Black lines represent the approximate length of the hypertrophic zone. Treatment with 2 µg/ml Shh inhibited hypertrophic differentiation and matrix mineralization (B). The overall length of the bone was not affected by treatment with Shh. (C,D) Histology. Sections from control (C) and Shh-treated (D) embryonic metatarsal rudiments were stained with Hematoxylin and Eosin. Zones of round periarticular proliferating (RP), flat proliferating (FP) and hypertrophic (HZ) cartilage were clearly demarcated. The fraction of cartilage containing hypertrophic chondrocytes was reduced in Shh-treated cultures, while the zone of proliferative chondrocytes was increased relative to controls. (E,F) Col10a1 expression. Expression of Col10a1, a marker of hypertrophic cartilage, was detected using in situ hybridization. The fraction of cartilage synthesizing Col10a1 mRNA was reduced in Shh-treated cultures (F) relative to untreated controls (E). Only dark field images are shown. Hybridization is seen as the bright white grains on the dark background. (G,H) PTHrP expression. Pthrp mRNA was localized using in situ hybridization. Little to no Pthrp mRNA was detected in untreated bone rudiments (E). Pthrp mRNA was detected in the periarticular cartilage (arrowhead) and in the perichondrium (arrow) of rudiments after treatment with Shh (H). Dark field and bright field images were merged using Photoshop software. Hybridization is seen as bright white grains on the gray background. Scale bar: 290 µm.

View larger version (125K): [in a new window]   Fig. 2. Effects of Shh on the expression of Tgfb1, Tgfb2 and Tgfb3 mRNA. The expression patterns for Tgfb1 (A,B), Tgfb2 (C,D) and Tgfb3 (E,F) mRNAs were determined by in situ hybridization for bone rudiments grown for 5 days in either 100% control conditioned medium (A,C,E) or 100% Shh-conditioned medium (B,D,F). Tgfb1 mRNA expression was detected in a subset of prehypertrophic and hypertrophic chondrocytes in untreated (A) and Shh-treated cultures (B). Tgfb2 transcripts were located in the perichondrium in untreated cultures (C). Treatment with Shh resulted in an increase in hybridization to the Tgfb2 probe in the inner and outer layers of the perichondrium (D). In untreated cultures, Tgfb3 expression was primarily detected in the perichondrium (E). Treatment with Shh resulted in an increase in Tgfb3 mRNA levels in the outer layers of the perichondrium (F). Both bright field (A-F) and dark field (A'-F') images are shown. Scale bar: 330 µm.

View larger version (86K): [in a new window]   Fig. 3. Localization of Ptc1 expression. Metatarsal bones from PtclacZ/+ mice were placed in culture and were either left untreated (A,B) or treated with 2 µg/ml Shh (C, D) for 72 hours. Bones were then stained with X-gal to localize Ptc1 expression. Both whole-mount, cleared cultures (A,C) and cryosections of stained tissue (B,D) are shown. The Ptc1 expression domain appears blue. In untreated cultures, light staining is observed in the perichondrium adjacent to the previously reported Ihh expression domain (B; small black arrows) as well as in flat, prehypertrophic chondrocytes. Shh-treated cultures demonstrate intense blue staining in all the perichondrium surrounding the entire bone (C,D; large black arrow) as well as variable staining in chondrocytes. No staining was detected in cultures from Ptc1+/+ mice (not shown).

View larger version (70K): [in a new window]   Fig. 4. Role of the perichondrium in signaling by Shh. Morphology of metatarsal cultures (A-D). Bone rudiments in which the perichondrium remained intact (A,B) or was stripped from the length of the rudiment (C,D) were either left untreated (A,C) or treated with 2 µg/ml Shh (B,D) for 5 days. A marked decrease in the length of the hypertrophic zone (black line) was observed in intact cultures treated with Shh when compared with untreated, intact controls. No difference in length of the hypertrophic zone was detected in Shh-treated, perichondrium-free cultures when compared with untreated, perichondrium-free cultures. (E-H) Col10a1 expression. Intact (E,F) and perichondrium-free rudiments (G,H) were either left untreated (E,G) or treated with 2 µg/ml Shh (F,H) for 5 days. The area of cartilage expressing Col10a1 was reduced in intact cultures treated with Shh when compared with the intact untreated controls. The area of cartilage expressing Col10a1 was not altered in stripped rudiments treated with Shh when compared with the untreated, stripped controls. Dark field images are shown. Hybridization is detected as bright white grains on the dark background. Scale bars: 360 µm in A-D; 250 µm in E-H.

View larger version (51K): [in a new window]   Fig. 5. Infection of embryonic metatarsal bone rudiments with adenovirus. Metatarsal rudiments infected with adenovirus that express the ß-gal reporter gene (A,C) or a dominant-negative mutation of the TGFß type II receptor (B,D) in the perichondrium were untreated (A,C), treated with TGFß (1 or 10 ng/ml; B) or Shh (2 µg/ml; D) for 5 days. Treatment with TGFß1 in rudiments infected with ß-gal virus resulted in a decrease in the overall length of the metatarsal bones and in a decrease in the length of the hypertrophic zone (black lines) when compared with untreated cultures. (A) The effects of TGFß1 were dose dependent. (B) TGFß1 treatment did not affect the overall length of the metatarsal or the length of the hypertrophic zone (black line) in rudiments previously infected with DNIIR virus. (C) Shh treatment of rudiments infected with the ß-gal virus resulted in a decrease in the length of the hypertrophic zone (black line). (D) Bone rudiments infected with DNIIR virus and treated with Shh did not show any decrease of the length of the hypertrophic area when compared with untreated rudiments. Metatarsal bones infected with an adenovirus that express the ß-gal reporter gene were used to determine the efficiency and location of infected cells. (E) Whole bone rudiments were stained for ß-galactosidase activity with X-gal. Infected cells appear blue. (F) Adenovirus infection was also visualized by whole-mount immunocytochemistry with a fluorescence-conjugated antibody directed to the adenovirus coat protein. Infected cells appear green. Staining only in the plane of focus is visible resulting in dark areas at either end of the bone. Scale bars: 250 µm in A-D; 180 µm in E,F.

View larger version (97K): [in a new window]   Fig. 6. Col10a1 and Pthrp expression in metatarsal bones infected with ß-gal or DNIIR adenovirus. Col10a1 (A-D) was localized by in situ hybridization to sections from bone rudiments infected with ß-gal virus (A,B) or DNIIR virus (C,D). Infected rudiments were either left untreated (A,C) or treated with 2 µg/ml Shh (B,D) for 5 days. Shh-treated bone rudiments infected with ß-gal virus showed a marked reduction of the fraction of cartilage expressing Col10a1. In those samples infected with DNIIR virus, Col10a1 expression was not affected after Shh treatment. Only dark field images are shown so that hybridization is seen as bright white grains on the dark background. Scale bar: 330 µm. In situ hybridization was used to localized Pthrp (E-H) mRNA in sections from bones that were infected with ß-gal (E,F) or DNIIR (G,H) adenovirus then either left untreated (E,G) or treated with Shh (F,H). Hybridization was detected in the perichondrium and periarticular region in bones infected with the control ß-gal virus and then treated with Shh. Little to no Pthrp mRNA was detected in untreated cultures or in cultures infected with DNIIR adenovirus and treated with Shh. Bright field (E-H) and dark field (E'-H') images are shown. Scale bar: 250 µm in E-H.

View larger version (85K): [in a new window]   Fig. 7. Col10a1 and Pthrp expression in Tgfb2- and Tgfb3-null cultures. Col10a1 (A-H) expression was localized by in situ hybridization to sections from metatarsal rudiments from Tgfb3-positive (A,B) and Tgfb3-null (C,D) littermates or Tgfb2-positive (E,F) and Tgfb2-null (G,H) littermates that were untreated (A,C,E,G) or treated with 2 µg/ml Shh (B,D,F,H) for 5 days. Col10a1 expression was reduced in Tgfb3-positive and Tgfb3-null cultures after treatment with Shh (A-D). Shh treatment also reduced the expression domain of Col10a1 in Tgfb2-positive bone rudiments; however, there was no reduction in Col10a1 expression in bone rudiments from Tgfb-null mice after treatment with Shh (E-H). Only dark field images are shown for Col10a1 in situ hybridization. Pthrp (I-L) expression was localized using in situ hybridization to sections from metatarsal cultures derived from Tgfb3-positive (I,J) and Tgfb2-null (K,L) embryos. Cultures were either left untreated (I,K) or were treated with 2 µg/ml Shh (J,L) for 5 days. As observed previously, Pthrp mRNA was detected in the periarticular region, the perichondrium and a subset of hypertrophic cells in Tgfb2-positive cultures that had been treated with Shh (J). Little to no hybridization was detected in untreated cultures from Tgfb2-positive and -null embryos (I,K), as well as in cultures from Tgfb2-null embryos treated with Shh (L). Both bright field (I-L) and dark field (I'-L') images are shown for PTHrP in situ hybridization. Scale bar: 330 µm.