Perichondrial expression of Wdr5 regulates chondrocyte proliferation and differentiation

Abstract

Wdr5 is developmentally expressed in osteoblasts and is required for osteoblast differentiation. Mice overexpressing Wdr5 under the control of the mouse alpha(1)I collagen promoter (Col I-Wdr5) display accelerated osteoblast differentiation as well as accelerated chondrocyte differentiation, suggesting that overexpression of Wdr5 in osteoblasts affects chondrocyte differentiation. To elucidate the molecular mechanism by which overexpression of Wdr5 in the perichondrium regulates chondrocyte differentiation, studies were undertaken using skeletal elements and cultured metatarsals isolated from wild-type and Col I-Wdr5 embryos. FGF18 mRNA levels were decreased in Col I-Wdr5 humeri. Furthermore, local delivery of FGF18 to the bone collar of ex vivo cultures of metatarsals attenuated the chondrocyte phenotype of the Col I-Wdr5 metatarsals. Impairing local FGF action in wild-type metatarsals resulted in a chondrocyte phenotype analogous to that of Col I-Wdr5 metatarsals implicating impaired FGF action as the cause of the phenotype observed. The expression of Twist-1, which regulates chondrocyte differentiation, was increased in Col I-Wdr5 humeri. Chromatin immunoprecipitation analyses demonstrated that Wdr5 is recruited to the Twist-1 promoter. These findings support a model in which overexpression of Wdr5 in the perichondrium promotes chondrocyte differentiation by modulating the expression of Twist-1 and FGF18.

Figure 1. The Col I-Wdr5 transgene accelerates chondrocyte differentiation

A): Humeri isolated from E14.5 wild-type (wt) and Col I-Wdr5 embryos, B) Paws isolated from E15.5 wild-type (wt) and Col I-Wdr5 embryos, C): metatarsals isolated from E15.5 wild-type (wt) and Col I-Wdr5 embryos and cultured for 4 days. Brackets indicate the expansion of the hypertrophic layer. Representative skeletal elements from at least three wild-type and Col I-Wdr5 embryos are shown.

Figure 2. Molecular analyses of chondrocyte differentiation

Sections of humeri and metatarsal cultures isolated from wild-type (wt) and Col I-Wdr5 embryos. Type II collagen (Col II), type X collagen (Col X) and osteopontin (OP) antisense riboprobes were digoxigenin (DIG-) labeled or radiolabeled with ³⁵S-UTP to a specific activity of at least 10⁸ cpm/μg of template. Representative skeletal elements from at least three wild-type (wt) and Col I-Wdr5 embryos are shown.

Figure 3. Wdr5 regulates FGF18 expression

A) In situ hybridization analysis for FGF18. Sections of humeri isolated from E14.5 wild-type (wt) and Col I-Wdr5 embryos are shown. The FGF18 antisense riboprobe was radiolabeled with ³⁵S-UTP to a specific activity of at least 10⁸ cpm/μg of template. Representative skeletal elements from at least three wild-type and Col I-Wdr5 embryos are shown. Arrows point to FGF18 expression. B) Quantitative Real-Time RT-PCR was performed on mRNA isolated from E14.5 wild-type (wt) (open bars) and Col I-Wdr5 (black bars) humeri. Expression of FGF18 mRNA was normalized to that of actin in the same sample. Values are expressed as the relative expression of the normalized mRNAs levels of the Col I-Wdr5 humeri versus that of wild-type (wt) humeri. Data shown are based on nine independent mRNA isolations ± S.E.M. * = p< 0.05 by Student t-test.

Figure 4. Local delivery of FGF18 to the perichondrium attenuates the chondrocyte phenotype of the Col I-Wdr5 embryos

Heparin acrylic beads incubated with 1 mg/ml FGF18 or BSA were implanted into the perichondrium of metatarsals isolated from E15.5 Col I-Wdr5 embryos (A) and their wild-type (wt) littermates (B). Metatarsals were then cultured for 4 days. Histological analysis and in situ hybridization analysis for Col X and OP were then performed. Arrows indicate heparin acrylic beads present in some sections. Representative metatarsal cultures from at least six wild-type (wt) and Col I-Wdr5 embryos are shown.

Figure 5. Blocking FGF signaling in the perichondrium recapitulates the Col I-Wdr5 phenotype in metatarsal cultures

A) Affigel blue beads incubated with10 nM or100 nM of FGF inhibitor or DMSO were implanted into the perichondrium of metatarsals isolated from E15.5 wild-type (wt) and Col I-Wdr5 embryos. Metatarsals were then cultured for 4 days. Histological analyses, using Toluidine Blue staining were then performed. B) In situ hybridization analyses for Col X and OP were performed on metatarsals isolated from E15.5 wild-type (wt) and Col I-Wdr5 embryos implanted with Affigel blue beads incubated with 100 nM of FGF inhibitor or DMSO. Representative metatarsal cultures from at least six wild-type (wt) and Col I-Wdr5 embryos are shown. Arrows point to beads present in some sections.

Figure 6. Wdr5 regulates Twist-1 expression

A) In situ hybridization analysis for Twist-1. Sections of humeri isolated from E14.5 wild-type (wt) and Col I-Wdr5 embryos are shown. Twist-1 antisense riboprobe was radiolabeled with ³⁵S-UTP to a specific activity of at least 10⁸ cpm/μg of template. Representative humeri from at least three wild-type (wt) and Col I-Wdr5 embryos are shown. Arrows point to the expression of Twist-1. B) Quantitative Real-Time RT-PCR was performed on mRNA isolated from E14.5 wild-type (wt) (open bars) and Col I-Wdr5 (black bars) humeri. Expression of Twist mRNA was normalized to that of actin in the same sample. Values are expressed as the relative expression of the normalized mRNAs levels of the humeri from Col I-Wdr5 embryos versus that of wild-type (wt) humeri. Data shown are based on nine independent mRNA isolations ± S.E.M. * = p< 0.05 by Student t-test. C) Quantitative ChIP analyses demonstrating a specific interaction of Wdr5 with the Twist-1 promoter. Values are shown as enrichment of the promoter sequences normalized for coding region sequences versus that obtained for input samples. Data shown are representative of that obtained with three independent experiments ± S.E.M. *= p<0.05 by Student t-test for ChIP samples versus input samples.