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Figures
- Fig. 1.
Pcdo is a nonsense allele of Spag17. (A) The Spag17 gene has 49 exons, and the position of the c.5236A>T ENU variant in exon 36 is indicated with red arrows (K1746* in the protein). Mouse SPAG17 has 2320 amino acids, and epitopes for the N-terminal and C-terminal SPAG17 antibodies are indicated. Blue arrows in the transcript indicate positions of the primers used in C. The mouse SPAG17 has coiled coil (blue), lysine-rich (blue dots), glycine-rich (red) and Pfam (gray) domains. (B) Sanger sequencing showing the A>T allele change in the Spag17Pcdo/Pcdo mutants. (C) Semi-quantitative RT-PCR of the Spag17 exon 33-39 and β-actin loading control. (D) Quantification of Spag17 expression in the testis, lung and brain tissues (relative to β-actin loading and normalized to testis wild-type levels: n=3 animals for each genotype; colors show littermates). (E) Western blotting with two different antibodies shows a lack of SPAG17 protein in Pcdo mutant testis compared with wild type and a decrease in the brain. (F) Analysis of the adult testis highlights another form of SPAG17 that is missing in the Pcdo mutants (red asterisks denote the loss or the reduction of SPAG17 isoforms in the mutant tissue).
- Fig. 2.
Spag17Pcdo does not interfere with skeletal development or primary ciliogenesis. (A-F) Alizarin Red- and Alcian Blue-stained adult mouse upper limb skeleton (A,B), femur (C,D) and tibia (E,F) from control (A,C,E) and Pcdo mutants (B,D,F). (G) The lengths of the humerus, radius, ulna, femur and tibia are shown; *P=0.017 (# indicates no significant difference; n=16 and 11 for Spag17control and Spag17Pcdo/Pcdo, respectively). (H,I) Immunocytochemistry images of the Spag17Pcdo/+ and Spag17Pcdo/Pcdo MEFs stained with ARL13B (green) and DAPI (blue). (J) The percentage of ciliated MEFs was not different between Spag17Pcdo/+ and Spag17Pcdo/Pcdo cells (n=4 animals for each genotype). Scatter plot in bars shows the mean and standard deviation; colors show littermates. Scale bars: 5 mm in A-F; 10 µm in I.
- Fig. 3.
Hydrocephalus was the first obvious phenotype in the Spag17Pcdo mutant line. (A-F) Coronal brain sections from Spag17wt/wt and Spag17Pcdo/Pcdo littermates, showing the dilated lateral ventricles at P7 and older in the mutants. (G) Quantification is shown for multiple sections of three or more animals for each stage and genotype. (H,I) Representative kymographs from Spag17control (H) and Spag17Pcdo/Pcdo (I). (J) Scatter plots of the frequency of the motile cilia beating measurements obtained from lateral ventricle and aqueductal cilia. Lateral ventricle cilia are hyperkinetic and beat with a rhythm significantly faster than in the Spag17 control animals (n=37 Spag17wt/wt lateral ventricle cilia and 69 Spag17Pcdo/Pcdo lateral ventricle mutant cilia obtained from three animals of each genotype; aqueduct, n=19 wild-type and 39 mutant cilia obtained from two wild-type and four mutant animals). (K) Scatter plots showing that there was no significant difference between flow speed of the flourescent microbeads in lateral ventricle brain slices (n=4 slices from three animals of each genotype). Scale bars: 500 µm in B,D; 1 mm in F; 2 µm in H,I.
- Fig. 4.
Aqueduct stenosis led to disturbed CSF flow and hydrocephalus in the Spag17Pcdo/Pcdo mice. (A,B) Hematoxylin and Eosin-stained coronal sections through the P3 aqueduct from Spag17wt/wt (A) and Spag17Pcdo/Pcdo (B). (C-F) Boxed areas of magnified aqueduct lumen (C,D) and SCO (E,F), as indicated in A,B. (G-J) Coronal P120 aqueduct sections from Spag17wt/wt (G) and Spag17Pcdo/Pcdo (H); boxed areas are magnified in I,J. Scale bars: 500 µm in A,B,G,H; 100 µm in C-F,I,J. (K) Scatter plots showing the speed of the moving beads inside the aqueduct (data collected from approximately four slices from two control and four mutant animals).
- Fig. 5.
Spag17Pcdo/Pcdo mutants are generally viable and develop PCD phenotypes. (A-D) Histological analysis of Spag17wt/wt (A,B) and Spag17Pcdo/Pcdo (C,D) trachea and lung showing mucus accumulation (asterisks) in the trachea and main bronchus in the mutant animals at P8 (A,C) and P14 (B,D). (E-J) Seminiferous tubules of the testis at 5 weeks (E,H), 10 weeks (F,I) and 4 months (G,J) from Spag17wt/wt (E-G) and Spag17Pcdo/Pcdo mutants (H-J). In E-G, arrows in the Spag17wt/wt sections point to a mature sperm flagellum. The Spag17Pcdo/Pcdo mutants lack a sperm flagellum at all stages presented, indicated by asterisks in H-J. Scale bars: 200 µm in A-D; 20 µm in E-J.
- Fig. 6.
SPAG17 is essential for development of the sperm flagellum but not for initiation of motile ciliogenesis in the brain or the respiratory tract. (A-D) Forebrain ependymal (A,B) and tracheal epithelial (C,D) cells of Spag17control (A,C) and Spag17Pcdo/Pcdo (B,D) animals stained for the ciliary axoneme with acetylated α-tubulin (red) and DAPI to stain nuclei (blue). (E-H) SEM images of the lateral wall (E,F) and medial wall (G,H) of the P8 forebrain ependymal cilia. No clear differences are observed in the overall morphology of the ependymal or the respiratory motile cilia. (I-L) Seminiferous tubules of Spag17wt/wt (I,K) and Spag17Pcdo/Pcdo (J,L) animals stained for acetylated α-tubulin (red) and DAPI (blue). No staining of the cilia can be detected in the Spag17Pcdo/Pcdo mutants (J,L) compared with Spag17wt/wt (I,K). Scale bars: 10 µm in A-H; 20 µm in I-L.
- Fig. 7.
SPAG17 is essential for central pair development and stability in the respiratory epithelial cilia. (A-C) TEM cross-section of the ependymal cilia from Spag17wt/wt (A) and Spag17Pcdo/Pcdo (B,C) showing no clear ultrastructural defects in the mutant ependymal cilia (n=58 cilia from wild-type and n=64 cilia from mutant cross-sections; three different specimens for each). (D-K) Higher magnification of the respiratory cilia axoneme cross-sections, showing lack of (arrows in F,G) or abnormal (arrows in J,K) C1 microtubule structure of the central pair apparatus in the Spag17Pcdo/Pcdo mutants (F,G,J,K; n=2 animals) compared with cross-sections of wild-type (D,E,H,I; n=2 animals) respiratory cilia. (L,M) TEM images of the respiratory epithelium of Spag17wt/wt (L) and Spag17Pcdo/Pcdo (M) animals (red lines indicate the axis of orientation of the central pair; the Spag17Pcdo/Pcdo mutant central pair(s) were abnormally oriented in different directions; thin white arrows in M point to cross-sections of cilia that have no C1 microtubule). (N,O) Basal processes of the basal bodies were normally and equally aligned in the respiratory epithelial cells from Spag17wt/wt (N) and Spag17Pcdo/Pcdo (O) mutants (red arrows). Scale bars: 250 nm in C,K; 500 nm in L,M; 1 µm in N,O.
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