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Development 138 (17): 3791-3801
Kif7 promotes hedgehog signaling in growth plate chondrocytes by restricting the inhibitory function of Sufu
Shu-Hsuan C. Hsu1,2,
Xiaoyun Zhang1,
Chunying Yu1,
Zhu Juan Li1,4,
Jay S. Wunder3,
Chi-Chung Hui1,4, and
Benjamin A. Alman1,2,5,*
1 Program in Developmental & Stem Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
2 Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada.
3 Samuel Lunenfeld Research Institute and Department of Surgery, Mount Sinai Hospital, Toronto, Ontario, Canada.
4 Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
5 Division of Orthopaedic Surgery and Department of Surgery, University of Toronto, Toronto, Ontario, Canada.
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Fig. 1. Sufu is differentially expressed in the growth plate. (A,B) Expression of Sufu and Ihh in wild-type mouse growth plates at E18.5 assessed by qRT-PCR (AC/RC, articular chondrocytes/resting chondrocytes; PC/PRC, proliferating chondrocytes/prehypertrophic chondrocytes; HC, hypertrophic chondrocytes). Data are shown as means with 95% confidence intervals indicated (n=3). (C) Immunohistochemical analysis of E16.5 wild-type mouse growth plates showed that Sufu is highly expressed by in AC/RC and PC/PRC regions, and fewer than 10% of the HC are stained positive for Sufu. (D) A lack of immunohistochemical staining for Sufu was found in Col2a1-Cre;Sufuf/f growth plate, demonstrating efficient deletion of Sufu in chondrocytes. (E) Effective deletion of Sufu in growth plate chondrocytes was also verified using western analysis. (F) P10 Col2a1-Cre;Sufuf/f mutant mice showed significant reduction in body length compared with wild-type littermates. (G) Alcian Blue/Alizarin Red staining showed a delay in ossification in the P10 tibial bones and P0 lumbar vertebrae of the Col2a1-Cre;Sufuf/f mice. (H) Safranin O staining revealed an expansion of the proliferative zone (PZ), a decrease in the length of the hypertrophic zone (HZ) and a delayed secondary ossification (SOC) formation in the tibia of P10 Col2a1-Cre;Sufuf/f mice (above). An increased length of the PZ and a reduction of the HZ were also found in the P10 mutant vertebral growth plates, as revealed by Hematoxylin and Eosin staining (below). (I) Hematoxylin and Eosin staining revealed an expansion of the proliferative zone, which is represented by the length from the end of the tibia to the beginning of the hypertrophic zone (black bar), and a reduction of the hypertrophic zone (red bar) in the Sufu-deficient tibial growth plates compared with wild-type littermates at E16.5. Lower panels show magnified images of the hypertrophic zone (red bar) seen in the upper panel. See also Figs S1, S2 and S4 in the supplementary material.
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Fig. 2. Sufu negatively regulates growth plate chondrocyte differentiation. (A-C) Histological and immunohistochemical analysis of the E16.5 mouse tibia showed the length of the hypertrophic zone (red bar), which is characterized by Col10a1 staining, is reduced in Col2a1-Cre;Sufuf/f embryos compared with wild-type littermates. An increase in the size of the proliferative zone, which represented by the length from the end of the tibia to the beginning of the Col10a1 expression zone (black bar), was found in the Col2a1-Cre;Sufuf/f embryos. This phenotype resembles the phenotype displayed by deletion of Ptch1, another repressor of Hh signaling, although less severe. Data are shown as means with 95% confidence intervals indicated (n=3). *P<0.05. (D) Expression of Hh target genes Gli1, Ptch1 and Hhip1 was assessed by qRT-PCR. Data are shown as means with 95% confidence intervals indicated (n=3-6). *P<0.05. (E) Protein levels of Gli2, full-length Gli3 (Gli3FL) and repressor form of Gli3 (Gli3R) in wild-type and Sufu-deficient growth plates were assessed by western analysis. Actin was used as loading control. Sufu-deficient chondrocytes exhibited elevated Gli2 level and an increase in the Gli3FL:Gli3R ratio (*non-specific band). See also Fig. S3 in the supplementary material.
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Fig. 3. Kif7 inactivation resulted in opposite effects on chondrocyte proliferation and differentiation to those of Sufu inactivation. (A) Immunohistochemical analysis of the tibia of E16.5 wild-type embryos showed that Kif7 expressed at high levels in the AC/RC region, and its expression progressively decreases as chondrocytes differentiate. (B) Expression of Kif7 in wild-type mouse growth plates at E16.5 assessed by qRT-PCR. Data are shown as means with 95% confidence intervals as indicated (n=3). (C) Safranin O and Col10a1 staining revealed an expansion of the hypertrophic zone (red bar), and reduced length of the proliferative zone (black bar) in the Kif7 knockout mice (Kif7–/–) compared with wild-type littermates. (D) Alcian Blue/Alizarin Red staining of wild-type and Col2a1-Cre;Kif7f/f littermates at E18.5 and P10. Col2a1-Cre;Kif7f/f embryos exhibited no gross abnormalities in skeletal development. (E,F) An increase in the length of the hypertrophic zone and a reduction in the length of the proliferative zone were found in Kif7–/– and Col2a1-Cre;Kif7f/f mice compared with wild-type littermates. Data are shown as means with 95% confidence intervals indicated (n=3). *P<0.05. (G) A significant decrease in the percentage of proliferating cells was found in Kif7–/– and Col2a1-Cre;Kif7f/f growth plates, as measured by immunohistochemical staining for Ki67. Data are shown as means with 95% confidence intervals indicated (n=3). *P<0.05. See also Figs S3 and S4 in the supplementary material.
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Fig. 4. Increased level of Sufu-Gli complexes in Kif7-deficent chondrocytes. (A) Sufu protein levels in wild-type and Kif7-deficient growth plates were assessed by western analysis. Actin was used as loading control. (B) Sufu mRNA expression levels were found to be comparable to the wild-type controls. Data are shown as means with 95% confidence intervals indicated (n=3). (C,D) Western analysis of Sufu in wild-type and Kif7-deficient chondrocytes following 20 µM cycloheximide (CHX) treatment. (E,F) Western analysis of Sufu in wild-type and Kif7-deficient chondrocytes following 25 µM MG132 treatment. Actin was used as loading control. (G) Fluorescence micrographs of cilia from wild-type, Col2a1-Cre;Sufuf/f and Col2a1-Cre;Kif7f/f growth plate chondrocytes. Gli2 (ciliary tip localization of Gli2 indicated by an asterisk), Gli3 (ciliary tip localization of Gli3 indicated by double asterisks), Sufu (ciliary base localization of Sufu indicated by a yellow arrow; ciliary tip localization of Sufu indicated by a green arrow) and Kif7 (ciliary tip localization of Kif7 indicated by a white arrow) are detected in the green channel. Cilia are detected by staining against acetylated α-tubulin (red channel). Centrioles are detected by staining against  -tubulin (blue channel) to label the base of the cilia. (H-J) Twenty-five to 50 ciliated cells from the proliferating zone of wild-type, Sufu-deficient and Kif7-deficient growth plates were examined for Gli2, Gli3, Sufu and Kif7 ciliary localization. Data are shown as percentages. (K,L) Western analysis of total Gli2 protein and Gli2 protein immunoprecipitated (IP) with anti Sufu antibody from control (C), cyclopamine- (Cy), purmorphamine- (Pur) and Shh-treated primary chondrocyte cultures (asterisk indicates the rabbit IgG band). The level of total Gli2 is comparable in all samples. The level of Gli2 co-immunoprecipitated with Sufu is similar in cyclopamine-treated and control cells. Significantly less Gli2 co-immunoprecipitates with Sufu in purmorphamine- and Shh-treated chondrocyte cultures. Data are shown as means with 95% confidence intervals indicated. **P<0.01. See also Fig. S4 in the supplementary material.
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Fig. 5. A dual function for Kif7 in Hh signaling modulation. (A-C) Histological and immunohistochemical analysis of the mouse tibial growth plates. Upper panels show Alcian Blue staining on E16.5 tibial sections; lower panels show Col10a1 immunostaining on E18.5 tibial sections (black bar, proliferative zone; red bar, hypertrophic zone). Data are shown as means with 95% confidence intervals indicated (n=3). *P<0.05. (D) Expression of Gli1, Ptch1 and Hhip1 in mouse growth plates of various mutants at E16.5 assessed by qRT-PCR. Data are shown as means with 95% confidence intervals indicated (n=3-6). *P<0.05. See also Fig S3 in the supplementary material.
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Fig. 6. Proposed model for how Sufu and Kif7 regulate Hh signaling in growth plate chondrocytes. (A) Schematic representation of Hh signaling regulation in chondrocytes. (a) In a wild-type cell, Sufu acts as a repressor in controlling Hh pathway activity. Kif7 possesses dual functions in regulating Gli transcription activity. (b) In the absence of Sufu, Kif7 acts as a repressor, resulting in a submaximal level of Hh pathway activation. (c) Loss of Kif7 results in increased Sufu activity, resulting in reduced Hh pathway activity. (d) Further augmentation of the Hh pathway activity is found in the absence of both Sufu and Kif7. (B) Schematic representation of Kif7-mediated dissociation of Sufu-Gli protein complexes at the primary cilium. Kif7 plays a functional role at the tip of the primary cilium in growth plate chondrocytes; it positively regulates Hh signaling activity via promoting the dissociation of Sufu-Gli complexes, leading to Gli-mediated transcriptional activation. Kif7 also function negatively in Hh signaling possibly through binding with Gli proteins in the cytoplasm.
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