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PTHrP and Indian hedgehog control differentiation of growth plate chondrocytes at multiple steps

Tatsuya Kobayashi¹, Ung-il Chung¹, Ernestina Schipani¹, Michael Starbuck², Gerard Karsenty², Takenobu Katagiri^1,, Dale L. Goad^1,, Beate Lanske^1, and Henry M. Kronenberg^1,*

¹ Endocrine Unit, Massachusetts General Hospital, Boston, MA 02114, USA
² Department of Human and Molecular Genetics, Baylor College of Medicine, Houston, TX 77030, USA
Present address: Department of Biochemistry, School of Dentistry, Showa University, Japan
Present address: Department of Pathology Angell Memorial Animal Hospital, Boston, MA, USA
Present address: Department of Oral Biology, Forsyth Institute, Boston, MA, USA

View larger version (34K): [in a new window]   Fig. 1. Generation of floxed and d allele of the Ppr locus. (A) Schematic representation of the Ppr locus, the targeting vector used for floxed allele generation and the floxed (fl) locus. Exons and selectable genes are indicated by gray and white boxes, respectively. loxP sequences are indicated by open triangles. Neo and tk cassettes are indicated as Neo and TK, respectively. Restriction enzyme sites for BamHI, KpnI, HindIII, RsrII, XhoI and XbaI are indicated as B, K, H, Rs, X and Xb, respectively. Probes used for Southern blot analysis are indicated with bold lines above the corresponding sequences. (B) In situ hybridization of PPR in 3-week-old mouse growth plates. (C) Northern blot analysis of PPR mRNA expression in the kidney of 8-week-old mice. (D-K) Southern blot analysis for the characterization of the Ppr d allele. DNA (10 µg) purified from wild type (+/+), homozygous floxed (fl/fl) and homozygous damaged (d/d) mice was digested with indicated restriction enzymes and hybridized with the probes indicated. Probes A and B are external probes in the 5'- and 3'-flanking regions of the targeted sequence, respectively. Probes C and S are exonic sequences for exon E3 and exon S of the Ppr gene, respectively. Probes Neo and TK are specific to the neo and the tk genes, respectively. (D) Only a single 8.5 kb band produced by the homologous recombination at the 5'-end of the targeting vector is seen in both (fl/fl) and (d/d). Note the band intensity of (d/d) is greater than that of (fl/fl), suggesting amplification of the 5'-flanking region. (E) Homologous recombination at the 3'-end was confirmed by an external probe B. Because the band intensities of the (d/d) and (fl/fl) are similar, we conclude that the 3'-flanking region is not amplified. The band in +/+ is not visualized probably because of the large size of the fragment (>18 kb). (F) An extra band for at 6.5 kb is present in d/d, in addition to the expected 8 kb band also seen in (fl/fl). (G) A band for tk is present only in d/d. (H) The band intensity for exon E3 is the same among in +/+, fl/fl and d/d. (I) The band intensity for S exon is about three times as intense in d/d as in +/+ or fl/fl. (J) Hybridization with Neo probe after RsrII digestion visualized two bands at 20 kb and 25 kb only in d/d. These may represent tandemly repeated sequences because one of the RsrII bands detected by Neo probe was also detected by probe D, corresponding to the broken bracket marked with # in L (data not shown). Neither of these bands was detected by TK probe (data not shown). (K) Each blot shown above was reprobed with the GAPDH probe for normalization. A representative blot is shown. (L) A possible structural model for the d allele of the Ppr locus. The d allele is likely to contain three copies of the targeting vector. Targeting vectors are integrated into the genome replacing the endogenous sequence of the corresponding region. One copy of tk sequence is present. A part of the 5'-flanking sequence including exon S is amplified with the targeting vector. Rs*, unmapped RsrII sites. Broken brackets, possible DNA fragments visualized in H. #, the probable fragment that hybridized with both Neo and D probes in H.

View larger version (67K): [in a new window]   Fig. 2. Cartilage-specific PPR ablation. (A) Tissue-specific activity of Cre recombinase. A Col2-Cre:R26R doubly transgenic, E15.5 embryo was stained with X-gal to visualize Cre activity. As well as chondrocytes, a part of perichondrium, ligaments, tendon and intra-joint tissues are stained blue representing lacZ activity. (B-K) Tibia of E17.5 embryos of homozygous floxed mice with (C,E,G,I,K) or without (B,D,F,H,J) Col2-Cre gene. (B-E) Bright field views of Hematoxylin and Eosin stained sections. (F-K) Dark field views of in situ hybridization with type II collagen (F,G), type X collagen (H,I) and PPR detected by a specific probe for the E1 exon (J,K). (L,M) The sterna of E 16.5 day-old embryos at the third inter-costal space of Pprfl/fl (L) and Col2-Cre:Pprfl/fl(M). The sternum of the Col2-Cre:Pprfl/fl mice is mostly occupied with hypertrophic chondrocytes.

View larger version (88K): [in a new window]   Fig. 3. Growth plates of Pprd/– mice. (A) Tibia of E17.5 Ppr+/+, Pprd/– and Col2-Cre:Pprfl/fl embryos. The growth plates and the bone length (indicated by black brackets) in the Pprd/– are greater than in the wild type, whereas they are reduced in the Col2-Cre:Pprfl/fl mouse. Note that the post-proliferating region (the hypertrophic zone and the marrow region that replaced hypertrophic chondrocytes) indicated by the blue brackets is not much different among these animals. (B) Differentiation marker expression in E17.5 embryos. Col-II, type II collagen; Col-X, type X collagen; Ihh, Indian hedgehog; Ppr, parathyroid hormone receptor (detected by R15B probe); Opn, osteopontin. (C) High-magnification view of growth plates of the E17.5 proximal tibia. In contrast to the great expansion of the hypertrophic region, the periarticular and columnar regions are slightly smaller in the Pprd/–. Brackets indicate the three regions: P, periarticular region; C, columnar region; H, hypertrophic region. (D) CaPpr expression overcomes the Pprd/– phenotype. In situ hybridization of the indicated markers shows the identical patterns between caPpr and caPpr: Pprd/– double mutants.

View larger version (75K): [in a new window]   Fig. 4. BrdU labeling analysis. (A) BrdU-positive chondrocytes of E17.5 embryos were counted in the area indicated by red and black lines for the periarticular region and the columnar region, respectively. Insets are magnified views of the periarticular (red outline) and columnar (black outline) regions. The number of BrdU-positive cells was divided by the number of nuclei to calculate proliferation rate (shown on the right). At least nine sections from three animals for each genotype were counted. *, statistically significant by ANOVA with P<0.05. (B) BrdU pulse-chase assay. Representative pictures of proximal tibias of mice sacrificed 24 hours after single BrdU injection at E17.5. Hypertrophic cells were identified by morphological criteria. The blue lines indicating the beginning of the hypertrophic zone were drawn by connecting the points at which bone collars started to form. The distance between the lines and the furthest BrdU-positive hypertrophic cells (indicated by red arrowheads), as well as number of BrdU-positive hypertrophic chondrocytes were measured and counted (shown on the right). Insets are magnified views of BrdU-positive hypertrophic cells. The distance was 32% greater in Pprd/– than in controls. The absolute number of BrdU-positive cells was increased in Pprd/– by 2. At least ten sections from two independent animals for each genotype were subjected to the analysis. * and **, statistically significant by ANOVA with P<0.05 and P<0.01, respectively. (C) BrdU labeling ratio was similarly calculated in the periarticular chondrocytes of E17.5 mice with cartilage-specific PPR ablation (shown on the right). Insets are magnified views of the periarticular region. At least nine sections from three independent animals for each genotype were counted. *, statistically significant by ANOVA with P<0.05.

View larger version (117K): [in a new window]   Fig. 5. Ihh signaling activities in periarticular chondrocytes. In situ hybridization for Ihh (B,F,J), Ptc (C,G,K), Pthrp (D,H,L), Hematoxylin and Eosin (A,E,I). (A-D) Pprfl/fl (control), (E-H) Col2-Cre:Pprfl/fl (I-L) Pprd/–. Arrowheads indicate Ptc and Pprp expression in the periarticular region.

View larger version (110K): [in a new window]   Fig. 6. Mosaic ablation of PPR in chondrocytes. (A) Mosaic activity of Cre recombinase of Ost-Cre transgenic mice in the growth plate. Ost-Cre:R26R doubly transgenic, an E17.5 embryo was stained with X-gal. ß-gal activity was seen in about 30% of the columnar chondrocytes. ß-gal-positive cells are few in the periarticular region. (B-I) In situ hybridization of E17.5 proximal tibias of Pprfl/fl (B,D,F,H) and Ost-Cre:Pprfl/fl (C,E,G,I). (D,E) Ihh is expressed in the hypertrophic region as well as ectopically differentiated hypertrophic cells in the Ost-Cre:Pprfl/fl growth plate. (F,G) Expression of Ptc (F,G) and Pthrp (H,I) is upregulated in the periarticular region in the mutant growth plate (arrowheads). (J) BrdU labeling. Increased labeling ratio despite the small periarticular region in Ost-Cre:Pprfl/fl. The areas indicated by red lines were subjected to BrdU-positive cell counting. Insets are magnified views of the periarticular region. Nine sections from three animals of each genotype were counted. *P<0.01.

View larger version (34K): [in a new window]   Fig. 7. Proposed model for the regulation of chondrocyte differentiation by PPR and Ihh signaling. (A) PPR signaling in the columnar chondrocytes blocks terminal differentiation (arrow 2). PPR signaling in chondrocytes also negatively controls the differentiation of periarticular chondrocytes to columnar chondrocytes (arrow 1). (B) Application of the model to mice with PPR mutations. Acceleration of cell supply to the columnar region at step 1 in Pprd/– leads to an increase in hypertrophic cell production because of the partial restoration of a cell amplification step in the columnar layer. Acceleration of step 1 in Ppr–/– compensates for the loss of the cell amplification system in the columnar layer. Ihh activity in the periarticular region negatively correlates with PPR signaling in these models. (C) Integrated model of PTHrP/Ihh feedback loop. PTHrP signaling directly blocks premature hypertrophic differentiation (a). Ihh positively regulates PTHrP expression in the periarticular region (b). In the presence of PTHrP, chondrocytes form columns between the periarticular region and the Ihh-expressing domain (c). Ihh action probably stimulates early chondrocyte differentiation to increase chondrocyte population with higher proliferation rate (d).