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- Fig. 1.
Ccdc151 gene knockout leads to perinatal lethality in mice. (A) Schematic representation of the Ccdc151tm1a allele. The lacZ reporter gene is inserted into an intronic locus, following exon 1, replacing exons 2 and 3 of the Ccdc151 gene. (B) Schematic representation of the Ccdc151tm1b allele. The Ccdc151tm1b allele is generated from the Ccdc151tm1a allele by Cre-mediated recombination. Cre recombination deletes exons 2 and 3 of the Ccdc151 gene and the neo targeting cassette from the Ccdc151tm1a genomic locus. The Ccdc151tm1b allele is a knockout of the Ccdc151 gene and in the text is labeled as the Ccdc151− allele. Ex, exon; En2A SA, splice acceptor site; IRES, internal ribosomal entry site; lacZ, bacterial β-galactosidase reporter gene; pA, poly A; hBactP, human β-actin promoter; neo, neomycin resistance gene; FRT, FLP recombination sites; LoxP, Cre recombination sites. (C) Schematic representation of Ccdc151 mRNA. Primers for the RT-PCR analysis, used to demonstrate the ablation Ccdc151 gene expression, are indicated. (D) Loss of Ccdc151 wild-type (WT) mRNA in homozygous Ccdc151-knockout animals is demonstrated by RT-PCR. (E) Analysis of genotype distribution of animals obtained by breeding between heterozygous Ccdc151+/− mice. Mendelian ratio of WT, Ccdc151+/− and Ccdc151−/− animals is observed at postnatal day 10 (P10). (F) Ccdc151-knockout animals demonstrate severe growth retardation. Weight measurements were performed starting at P5. From P6, Ccdc151−/− animals weighed significantly less when compared to WT animals (mean±s.e.m.; unpaired t-test; *P<0.05; ***P<0.001; ****P<0.0001). (G) Photograph of a Ccdc151−/− animal and WT littermate. (H) Most of the Ccdc151−/− animals do not survive to weaning. Kaplan–Meier survival plot of the Ccdc151-null (N=10) and WT (N=10) animals; log-rank test (Chi square=13.13; d.f.=1; P=0.003).
- Fig. 2.
Ccdc151 is expressed in ependymal cells of the ventricular brain system in mice. (A) Dorsal and ventral views of the brains from Ccdc151+/− animals, expressing the Ccdc151-lacZ reporter gene (a,b), and wild-type animals (c,d). The whole-mount brains were stained with X-gal/FeCN and β-galactosidase expression was visualized by steriomicroscopy. Red dashed lines indicate the sections of the brain presented in panel B. (B) Histological analysis of Ccdc151 gene expression. Sections were obtained from the Ccdc151-lacZ brain presented in panel A and stained with eosin. Lv, lateral ventricle; d3v, dorsal third ventricle; 3v, third ventricle; Aq, aqueduct of Sylvius. Scale bars: 1 mm (A); 500 μm (B); 50 μm (B, inset).
- Fig. 3.
Three-dimensional microCT imaging of Ccdc151-lacZ reporter gene expression in intact mouse brain detects Ccdc151 expression in the ventricular brain system. (A) Representative microCT multiple intensity projection (MIP) image of the wild-type (WT) brain stained with X-gal/FeCN and scanned with the resolution of 7.9 µm/voxel. (B) Representative microCT MIP image of the Ccdc151-lacZ heterozygous brain stained with X-gal/FeCN and scanned with a 9 µm/voxel resolution. (C) Whole-mount heterozygous brain stained with X-gal/FeCN. (D) Alignment of microCT-derived 2D sections of the WT brain (a-c), microCT-derived sections of the heterozygous Ccdc151-lacZ brain (a′-c′) and 2D section derived from a virtually built 3D model obtained using Brain Explorer2 software defining only the ventricular compartment of the mouse brain, marked by green (a″-c″). The regions of increased microCT-detected density (white) in a′-c′ is in remarkable agreement with corresponding virtual 2D coronal sections of the mouse brain, with the ventricular system highlighted in green (a″-c″). (E) Screenshot of the transfer function editor windows of the CTvox analyzer (Bruker software) demonstrates setting of the RGB transfer function curves for building a color volume-rendered 3D model; color coded for the tissue density function: red-blue-green; transparency level defined by the purple line. (F) MicroCT color volume-rendered 3D model of the Ccdc151-lacZ brain stained with X-gal/FeCN demonstrates highest densities in the ventricular region of the mouse brain. (G) 3D model of the mouse brain, created using Brain Explorer2 software, defining only the ventricular compartment of the mouse brain (green).
- Fig. 4.
Ccdc151-knockout animals develop postnatal hydrocephalus. (A) Dorsal view of mouse brain from homozygous Ccdc151−/− animals stained with the X-gal/FeCN protocol. (B) MicroCT-derived transversal sections of the Ccdc151−/− brain demonstrate enlargement of the lateral ventricles. (C-F) Coronal sections of the Ccdc151−/− knockout brain; microCT-derived coronal sections of the forebrain (C,E) and corresponding histological sections (D,F) demonstrate dilation of lateral ventricles and cortex thinning. (G) Screenshot of the transfer function editor windows of the CTvox analyzer (Bruker software). Settings of the RGB transfer function curves for building a color volume-rendered 3D model, color coded for the tissue density increases: red-blue-green; transparency level defined by the purple line. (H) MicroCT color volume-rendered 3D model of the Ccdc151−/− knockout brains stained with X-gal/FeCN demonstrate highest densities in the ventricular region of the mouse brain. Ependymal cell integrity is interrupted in the dorsal part of the lateral ventricles. (I-L) Quantification of the pathological hydrocephalus of the murine brain of Ccdc151+/− (N=3) and Ccdc151−/− (N=3) at P12. (I) Measurements of the total brain volume; mean+s.e.m.; Mann–Whitney one-tailed t-test, *P=0.05. (J) Forebrain volume; mean+s.e.m.; Mann–Whitney one-tailed t-test, *P=0.05. (K) Ventricular volume; mean+s.e.m.; Mann–Whitney one-tailed t-test, *P=0.05. (L) Cerebellar volume; mean+s.e.m.; Mann–Whitney one-tailed t-test, P=0.09 (non-significant).
- Fig. 5.
Ccdc151-knockout animals develop communicating hydrocephalus. (A,B) MicroCT analysis of paraffin-embedded whole-mount brains imaged at a resolution of 7.9 µm voxel size. Virtual microCT-derived sagittal sections from the brain of Ccdc151−/− (A) and wild-type (WT) animals (B) demonstrate continuity of the aqueduct of Sylvius. (C,D) MicroCT imaging analysis of the brains treated with Lugol's solution as a contrasting agent. Brains were imaged at 6.9 µm voxel size resolution. Virtual microCT-derived sagittal sections of the Ccdc151−/− (C) and WT (D) brains. (E,F) Virtual dissection of the brains by placing transversal and sagittal planes across the paraffin-embedded brains from Ccdc151−/− (E) and WT (F) animals. Lv, lateral ventricle; d3v, dorsal third ventricle; 3v, third ventricle; 4v, fourth ventricle; Aq, aqueduct of Sylvius.
- Fig. 6.
MicroCT analysis of continuity of the aqueduct of Sylvius. (A,B) Virtual microCT-derived transversal sections from the brain treated with the Lugol contrasting agent and imaged with a resolution of 6.9 µm voxel size. The continuity of the aqueduct in the brain of Ccdc151−/− (A) and wild-type (WT; B) animals is indicated by an arrow. Sequence of sagittal sections demonstrating aqueduct continuity from the brain of Ccdc151−/− (a1-a5) and WT (b1-b5) animals. Arrows indicate aqueduct.
- Fig. 7.
Targeted deletion of Ccdc151 in mice leads to left-right asymmetry defect, a trait of the PCD disease. (A) Ccdc151-knockout animals presented with situs solitus (SS), situs inversus totalis (SIT) and situs inversus abdominalis (SIA). RV, right ventricle; LV, left ventricle; St, stomach; 1-5 (white numbers), numeration of lung lobes; 1-3 (black numbers), numeration of liver lobes. (B) Summary of the phenotypes observed in Ccdc151-knockout animals.
- Fig. 8.
The Ccdc151 gene is expressed in testes and its targeted deletion leads to defective spermatogenesis. (A) Whole-mount staining of testes from Ccdc151-lacZ animals and wild-type (WT) control using the X-gal/FeCN protocol, demonstrating expression of Ccdc151 in testicular seminiferous tubules of the testis. Scale bars: 500 μm. (B) Schematic representation of the conversion of the Ccdc151tm1a allele into the conditional allele Ccdc151tm1c upon FLP-dependent recombination. Ex, exon; En2ASA, splice acceptor site; IRES, internal ribosomal entry site; LacZ, bacterial β-galactosidase reporter gene; pA, poly A; hBactP, human β-actin promoter; neo, neomycin resistance gene; FRT, FLP recombination sites; LoxP, Cre recombination sites. (C) Schematic representation of ROSA26ERT2-Cre recombinase induction by tamoxifen injections. (D) Schematic representation of the Ccdc151 knockout allele, after induction of Cre recombination. (E,F) Characterization of spermatogenesis after induction by tamoxifen of Cre recombinase in Ccdc151Con/Con ERT2-Cre (N=3) and Ccdc151Con/+ ERT2Cre (N=3) animals: sperm count, mean+s.e.m.; Mann–Whitney t-test, P=0.06 (E) and % of sperm motility, mean+s.e.m.; Mann–Whitney t-test, *P=0.05 (F).
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