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Genetic manipulation of hedgehog signaling in the endochondral skeleton reveals a direct role in the regulation of chondrocyte proliferation

Fanxin Long, Xiaoyan M. Zhang^*, Seth Karp, Yingzi Yang and Andrew P. McMahon

Harvard University, Department of Molecular and Cellular Biology, Cambridge, MA 02138, USA
^* Present address: Curis, 45 Moulton Street, Cambridge, MA 02138, USA
Present address: Department of Surgery, Brigham and Womern’s Hospital, 75 Francis St, Boston, MA 02115, USA
Present address: Genetic Disease Research Branch, National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA

View larger version (42K): [in a new window]   Fig. 1. Conditional removal of Smo activity in chondrocytes. (A) Schematic of the wild-type Smo allele (Smowt) and the conditional allele (Smoc). Below is a representative Southern blot identifying the targeted allele. Following EcoRV digestion, the 5' probe detects bands of 13 kb and 9 kb from wild-type and Smoc alleles, respectively. After BamHI digestion, the 3' probe detects bands of 9.6 kb and 7.2 kb from wild-type and Smoc alleles, respectively. (B) Organization of the Col2-Cre transgene. G.S.S., ß-globin splicing sequence. Below are whole-mount X-gal staining of E12.5 embryos that carry each of the three independently generated Col2-Cre transgenes (15, 3 and 10), and a Rosa26-LacZ reporter allele that requires Cre-mediated recombination for lacZ expression (see text).

View larger version (20K): [in a new window]   Fig. 2. Removal of Smo reduces the growth of long bones. (A) A comparison of E18.5 limb skeletons of wild type, Smo conditional null and Ihhn/Ihhn embryos. FL, forelimb; HL, hindlimb. (B) Relative lengths of E18.5 long bones of Cre10; Smon/Smoc embryos compared with Ihhn/Ihhn and wild-type bones. T, tibia; F, fibula; U, ulna; H, humerus; S, scapula; n=7. (C) Growth of the limbs at postnatal day 3 (P3) and P9 in Cre10; Smon/Smoc (MT) versus wild-type (WT) mice.

View larger version (77K): [in a new window]   Fig. 3. Removal of Smo activity abolishes Hh signaling in the cartilage. In situ hybridization with 35S-labeled probes for Ptc1 and Gli1 of tibial sections of Smon/Smoc embryos that carry either Cre15 or Cre10 transgenes, compared with wild-type and Ihh mutant embryos at E14.5 and E18.5.

View larger version (78K): [in a new window]   Fig. 4. Histological examination of tibial development after removal of Hh signaling. Normal hypertrophy of chondrocytes in Cre10; Smon/Smoc embryos at E14.5 and E18.5. Boxed regions shown at a higher magnification to the right. By contrast, Ihh mutants show a delay in hypertrophy at E14.5, and apositional hypertrophy that extends close to the articular surface at E18.5.

View larger version (123K): [in a new window]   Fig. 5. Normal expression of markers for chondrocyte differentiation after conditional removal of Smo activity from chondrocytes. Expression of the indicated probes was examined by in situ hybridization (35S-labeled probes) in tibia sections at E14.5 and E18.5.

View larger version (89K): [in a new window]   Fig. 6. Removal of Smo activity in chondrocytes reduces chondrocyte proliferation. (A) Similar reduction in chondrocyte proliferation between Cre10; Smon/Smoc and Ihhn/Ihhn embryos. Proliferation was scored in tibal sections after in utero labeling of E14.5 embryos with BrdU. (B) In situ hybridization of a 35S-labeled Ptc1 probe to tibial sections of Cre10; Smon/Smoc embryos indicates a complete absence of Ihh signaling in proliferating chondrocytes at E13.5. Perichondrial signaling is unaffected. (C) Alcian Blue staining of the tibia at E13.5 indicates a similar reduction in growth between Cre10; Smon/Smoc and Ihhn/Ihhn embryos. (D) In situ hybridization (35S-labeled probes) of cyclin D1 on tibial sections of E14.5 embryos. Expression of Cyclin D1 is downregulated in chondrocytes of both Cre10; Smon/Smoc and Ihhn/Ihhn embryos.

View larger version (69K): [in a new window]   Fig. 7. Ectopic activation of Ihh signaling in chondrocytes. (A) Transgene constructs used to drive ectopic expression of chick Ihh or Smo* using a UAS-Gal4 system. The UAS constructs contain the basic promoter region and the poly A signal from the murine Wnt1 gene, and the insulator sequence from the chick ß-globin gene. (B,C) In situ hybridization of indicated 35S-labeled probes to tibial sections at E14.5 and E16.5 reveals ectopic Ihh signaling driven by chick Ihh (B) or Smo* transgene (C). Note that the chick Ihh probe reacts weakly with the endogenous murine Ihh in wild-type tibia. Expression of Smo* is detected by an alkaline phosphatase (AP) probe.

View larger version (42K): [in a new window]   Fig. 8. Promotion of chondrocyte proliferation by ectopic Ihh signaling. (A) BrdU labeling indices in tibial sections of indicated genotypes at E14.5. (B) BrdU labeling reveals enhanced chondrocyte proliferation at E16.5 following ectopic Ihh expression. (C) BrdU labeling indices in E16.5 embryos after ectopic expression of Ihh or Smo* reveals enhanced proliferation within zone 1, the zone with lower proliferative activity in wild-type embryos. H, hypertrophy zone; Whole, entire proliferative zone.