Fig 1.
Femur bone development is affected in Spag17-deficient mice.
(A) Alcian Blue/ Alizarin Red staining of femur from wild-type (+/+) and Spag17-mutant (-/-) mice. Measurement of femur length and calcified femur bone length shows Spag17-mutant limbs are shorter than wild-type mice. Scale bars, 0.5 mm. (B) Histological and morphometric studies on femur shows increase bone formation in mutant mice. Scale bars, 200 μm. (C) von Kossa staining reveals increased calcification in mutant mice. (D) Micro-CT structural analysis on femurs; mid-sagittal sections (left) and cross-sectional trans-axial views at the mid-diaphysis (right). Images were acquired in 600 projections in 180 degrees of rotation with 500 miliseconds exposure per projection. Scale bars, 200 μm. (E) mRNA expression of osterix (Osx) and osteocalcin (Ocn) measured by qPCR. (+/+), wild-type; (-/-), Spag17-mutant mice. Data are presented as means ± SEM from ≥7 mice. * Indicates statistically significant differences, p< 0.05. hz: hypertrophic zone; BA/TA: bone area/total area; MA/TA: marrow area/total area; BV/TV: bone volume/total volume; Osx: osterix; Ocn: osteocalcin.
Fig 2.
Tibia bone development is affected in Spag17-deficient mice.
(A) Alcian Blue/ Alizarin Red staining on tibia from wild-type (+/+) and Spag17-mutant (-/-) mice. Measurement of tibia length and calcified tibia bone length shows Spag17-mutant limbs are shorter than wild-type mice. Scale bars, 0.5 mm. (B) Histological and morphometric studies on tibia shows cartilage and bone defects. Scale bars, 200 μm. (C) von Kossa staining. (D) Micro-CT structural analysis on tibias; mid-sagittal sections (left) and cross-sectional trans-axial views at the mid-diaphysis (right). Images were acquired in 600 projections in 180 degrees of rotation with 500 miliseconds exposure per projection. Scale bars, 200 μm. (E) mRNA expression of osterix (Osx) and osteocalcin (Ocn) measured by qPCR. (+/+), wild-type; (-/-), Spag17-mutant mice. Data are presented as means ± SEM from ≥7 mice. * Indicates statistically significant differences, p< 0.05. hz: hypertrophic zone; BA/TA: bone area/total area; MA/TA: marrow area/total area; BV/TV: bone volume/total volume; Osx: osterix; Ocn: osteocalcin.
Fig 3.
Disrupted development of the sternum in Spag17-/- mice.
(A) Schematic representation of normal sternal bones (M, manubrium; S1 to S4, sternebrae 1 to 4; X, xiphoid). (B,C and D) Analysis of the sternum by micro-CT scanning shows fused sternebrae in the mutant mice. Pictures are representative images from respective supporting videos. (Fig 3B is representative of S1 Video; Fig 3C is representative of S2 Video; and Fig 3D is representative of S3 Video.) (E and G) Alcian blue/ Alizarin red staining. Failure of ribs 4, 5 and 6 to complete their attachment to the sternum and promote sternebrae separation is apparent in the stained preparations in the Spag17-/- mice (arrows). (F and H) Representative H&E staining shows the presence of normal cartilage development that prevents fusion of the vertebrae in wild type animals. (+/+), wild-type; (-/-), Spag17-mutant mice. * Fused sternebrae.
Fig 4.
Cranial and phalanges defects in Spag17-/-.
(A) Micro-CT imaging of mineralized skull. Note the demineralization of the skull in the mutant mouse (white arrows). (B and E) Quantitative mineralization measurement by micro-CT scanning. (C) Alkaline phosphatase activity in cultured calvarial osteoblast. (D) Micro-CT imaging of mineralized forelimb. Insert shows reduced metacarpal mineralization by Alcian Blue/ Alizarin Red staining. Micro-CT images were acquired in 360 projections by 360 degree rotation, with 680 millisecond exposure per projection. White and black arrows indicate less metacarpal mineralization in the mutant mouse. (+/+), wild-type; (-/-), Spag17-mutant mice. Data are presented as means ± SEM. * Indicates statistically significant differences, p< 0.05.
Fig 5.
Primary cilia are altered in Spag17-deficient osteoblast, chondrocyte and MEFs cells.
Cells were stained with anti-acetylated α-tubulin to visualize primary cilia and DAPI as a nuclei marker. (A) Primary cilia length from osteoblast cells. Scale bars, 5 μm. (B) Percentage of osteoblast cells expressing primary cilia. Scale bars, 20 μm (C) Primary cilia length from chondrocyte cells. Scale bars, 5 μm. (D) Percentage of chondrocyte cells expressing primary cilia. Scale bars, 20 μm. Mouse embryonic fibroblasts (MEFs) were isolated from embryos at E12.5. (E) Detection of Spag17 mRNA and protein in MEFs. RT-PCR products were generated by primers from exon 4 and 5. The knockout mice have a deletion of the entire exon 5. Detection of SPAG17 protein by western blot using an antibody against C-terminal domain. (F) After serum-starvation to promote cilia growth, primary cilia were visualized in the MEFs with acetylated tubulin antibody, and DAPI as a nuclei marker. Primary cilia from Spag17-mutant MEFs were significantly shorter than wild-type. Scale bars, 5 μm. (G) Spag17 knockdown in WT MEFs cells after treatment with Spag17 siRNA duplex reproduced the shorter primary cilia phenotype. Scale bars, 5 μm. Arrows indicate primary cilia. (+/+), wild-type, (-/-); Spag17-mutant mice. Data are presented as means ± SEM. * Indicates statistically significant differences, p< 0.05.