Fig 1.
Cfap53 is specifically expressed in cells with motile cilia.
Cfap53lacZ mouse embryos and adult tissues were stained with the β-galactosidase substrate X-gal. The images include lateral (A) and ventral (B) views of E8.0 embryos; a sagittal section of the brain at 3 weeks of age (C), with higher magnification views of the boxed regions being presented in (D) and (E); the trachea at 8 weeks of age (F), with an image for a non-transgenic (Tg-) also being shown; sagittal section of the trachea at 8 weeks of age (G), with a higher magnification view shown in (H); the fallopian tube at 6 weeks of age (I); a sagittal section of the fallopian tube (J); and ducts of the testis at 10 weeks of age (K), with a higher magnification view shown in (L). Scale bars, 2 mm (K), 500 μm (C, G, J, L), 50 μm (D, E), 20 μm (H).
Fig 2.
Laterality defects, hydrocephalus, and ciliary motion defects in Cfap53 mutant mice.
(A) Genetic structure of the WT mouse Cfap53 locus and the generation of two types of knockout alleles lacking either exon 2 or exons 2 to 8. (B) Smaller body size and development of hydrocephalus in Cfap53–/–mice at 4 weeks of age. (C) Laterality defects of Cfap53–/–mice. (D) Survival curve for Cfap53–/–mice (n = 14), with all animals dying by 6 weeks of age. (E) In situ hybridization analysis of Nodal expression in Cfap53+/–and Cfap53–/–mice at E8.0. Nodal expression was missing in the LPM of Cfap53–/–embryos. Arrowheads indicate the node. (F) Immotility of node cilia in Cfap53–/–embryos at E8.0. Red signals for the Cfap53+/–embryo indicate motion trajectory in the corresponding movie. The red signal for the Cfap53–/–embryo reflects the trajectory of a bubble. Dashed lines indicate the outline of the node. (G) Wave forms of tracheal and brain ventricle cilia determined from corresponding videos, S3 and S4 Videos. (H) Beat frequency for tracheal cilia of Cfap53–/–and control mice (dots and triangles indicate Cfap53+/+ and Cfap53-/- mice, respectively) determined at 25°C. Data are presented as mean ± SD (n = 18 independent experiments); two tailed Student’s t-test (*p = 0.0041). See also S3 Video.
Fig 3.
Differential localization of CFAP53 in tracheal and node cilia.
(A–L) Immunofluorescence staining of the node of WT (A-C, G-L) or Cfap53–/–(D-F) embryos at E8.0 with antibodies to CFAP53 (A, D, G, J), to acetylated (acet.) Tubulin (B and E), to PCM1 (H) and to γ-Tubulin (K). Scale bars, 10 μm (A-F). Higher magnification images are shown in insets. Scale bars, 5 μm (A-F). Note that CFAP53 is mainly localized at the base of nodal cilia (centriolar satellites positive for PCM1), and weakly in the axoneme. Scale bar, 2 μm (G-L). (M-R) Immunofluorescence analysis of an isolated tracheal MCC from a WT (M-O) or Cfap53–/–(P-R) mouse. CFAP53 is detected at the base of cilia (the centriolar satellites) and in the axoneme. Scale bar, 5 μm.
Fig 4.
Localization of DNAH5 in ciliated cells of the trachea and node.
(A-F) Immunofluorescence staining with antibodies to DNAH5 (green) and to acetylated (acet.) Tubulin (magenta) of the node at E8.0 and adult trachea (G-L) from Cfap53–/–and control mice. (D-F, J-L) DNAH5 was absent from node cilia but maintained in tracheal cilia of Cfap53–/–mice. Scale bars, 20 μm. Insets show higher magnification views. Scale bars, 2 μm.
Fig 5.
Localization of DNAH11 in ciliated cells of the trachea and node.
(A-F) Immunofluorescence staining with antibodies to GFP (green) and to acetylated (acet.) Tubulin (magenta) for the node at E8.0 and adult trachea (G-O) of Cfap53–/–; Dnah11Venus and control mice. (D-F, J-L) DNAH11::Venus was absent from node cilia but was maintained in tracheal cilia of Cfap53–/–mice. In (C) and (F), the arrowhead indicates a node cilium. Scale bars, 10 μm (A-F) or 5 μm (G-O). Insets represent higher magnification views.
Fig 6.
Phenotype of Dnah9 mutant mouse.
(A) The exon-intron structure of mouse Dnah9 gene and generation of knockout allele lacking exon 30. Note that the motor domains of DNAH9 are encoded by exons 29–32 and exons 34–35. (B) Eight pups generated by CRISPR-mediated mutagenesis were genotyped by PCR. (C) External appearance of the same eight pups whose genotype is shown in (B). Arrowheads denote the position of the stomach. Note that all the Dnah9-/- (KO) mice have the stomach at the normal position. (D) Laterality of visceral organs in WT and Dnah9-/- mice. The arrowheads indicate the position of the heart apex. (E) Dnah9–/–mice developed hydrocephalus (arrowhead). (F) Wave forms of tracheal and brain ventricle cilia of Dnah9+/+ and Dnah9–/–mice. See also S7–S10 Videos. (G) Beat frequency for tracheal cilia of Dnah9–/–and control mice. Data are presented as mean ± SD (n = 14 independent experiments); two tailed Student’s t-test (*p<0.05). See also S7 and S8 Videos.
Fig 7.
ODAs are completely lost in node cilia but largely maintained in tracheal cilia of Cfap53–/–mice.
(A–D) TEM of node cilia of Cfap53+/+ (A) and Cfap53–/–(C) embryos at E8.0. Higher magnification views of corresponding doublet microtubules indicated by the arrowheads are shown in (B) and (D), respectively, together with schematic diagrams of the microtubule pairs and ODAs (blue protrusions). Scale bars, 100 nm. (E–I) TEM of tracheal cilia of adult Cfap53+/+ (E) and Cfap53–/–(G) mice. Higher magnification views of corresponding doublet microtubules indicated by the arrowheads are shown in (F), (H), and (I) together with schematic diagrams of the microtubule pairs and ODAs. Scale bars, 100 nm. (J and K) Distribution of the number of ODAs per axoneme for node (J) and tracheal (K) cilia of Cfap53–/–and Cfap53+/+ mice determined from TEM images. (L) Nine peripheral doublet microtubules were numbered from DMT1-DMT9, on the base of their relative position to the central pair: DMT1 is the doublet microtubule located at the position perpendicular to the central pair microtubules. The frequency of ODA loss at each DMT of Cfap53–/–tracheal cilia is shown by a circular plot. Note that DMTs with the line indicated by asterisk (#3 and #8) are preferentially lost (ρ = 0.04263 by Rayleigh’s test). (M) Distribution of the number of ODAs per axoneme for tracheal cilia of Cfap53; Rsph4a double mutant mice determined from TEM images.
Fig 8.
Association of CFAP53 with axonemal microtubules of tracheal cilia as well as with DNAIC proteins and TTC25.
(A) Isolated axonemes of tracheal cilia were treated (or not, Cont) with 0.6 M NaCl or NaI and then centrifuged at 15000 × g for 12 min at 4°C, and the resulting precipitate and supernatant fractions were subjected to immunoblot analysis with antibodies to CFAP53, to DNAIC2 (ODA marker) and to α-Tubulin. (B–E) HEK293T cells transfected with expression vectors for HA-tagged CFAP53 or MYC epitope-tagged DNAIC1, DNAIC2, DYNLL1 (cytoplasmic dynein light chain), TTC25, CCDC114, or CCDC151 (or with the corresponding empty vectors), or GFP-tagged DNAH11 N-terminus fragments 1–1000 aa or 1001–1700 aa, as indicated, were subjected to immunoprecipitation (IP) with antibodies to HA or to MYC or to GFP, and the resulting precipitates as well as the original whole cell lysates (WCLs) were subjected to immunoblot analysis with antibodies to HA, MYC or GFP. CFAP53 interacted specifically with the axonemal dyneins DNAIC1, DNAIC2 and DNAH11 N-terminus and the DC member TTC25.
Fig 9.
Genetic interaction between Cfap53 and Ttc25 in node cilia.
(A–F) Immunofluorescence staining with antibodies to CFAP53 (green) and to acetylated (acet.) Tubulin (magenta) of the node from Ttc25–/–and control embryos at E8.0. CFAP53 was localized at the axoneme and base of node cilia of Ttc25–/–embryos. (G–L) Immunofluorescence staining with antibodies to TTC25 (green) and to acetylated (acet.) Tubulin (magenta) of the node from Cfap53–/–and control embryos at E8.0. TTC25 was lost from the axoneme of node cilia in the Cfap53-/-. Scale bars, 10 μm. Insets show higher magnification views. Scale bars, 2 μm.
Fig 10.
Differential role of CFAP53 in 9+0 and 9+2 motile cilia.
(A) Schematic diagram for the expression of ODA proteins (DNAH5, DNAH11, DNAH9) in node and tracheal cilia of Cfap53–/–and WT mice. All three dynein heavy chain proteins were maintained in tracheal cilia, whereas DNAH5 and DNAH11 were completely lost in node cilia of the mutant mice. DNAH9 was not detected in node cilia of WT or Cfap53–/–mouse embryos. DNAH5 and DNAH11 (or DNAH9) are indicated by blue color on the axonemes. (B) CFAP53 (53) interacts with TTC25 (25) at the centriolar satellites. It also interacts with ODA proteins including dynein heavy chains such as DNAH11 and possibly DNAH5/9. CFAP53 likely facilitates the transport of TTC25 and the dyneins into cilia. An unknown protein (X) that can also interact with TTC25 and ODA proteins and mediate their transport into the tracheal cilia in the absence of CFAP53 is either not expressed or is nonfunctional in node cells.