Ciliopathy-associated gene Cc2d2a promotes assembly of subdistal appendages on the mother centriole during cilia biogenesis

The loss of Cc2d2a in mouse leads to embryonic lethality

Three protein-coding transcript variants are produced from the Cc2d2a gene. To eliminate all transcripts, we replaced Cc2d2a exons 6 to 8, shared by all variants, with a targeting vector containing a β-gal reporter and a neomycin selection cassette (Fig.1a) through standard homologous recombination in ES cells. Southern blotting of genomic DNA from ES clones using an exon 5-6 probe (Fig. 1a) showed 12.7 and 9.9 kb EcoRI fragments for the wild type and Cc2d2a^-/- alleles, respectively (Fig. 1b, Supplementary Fig. 1). We confirmed that the embryos homozygous for the targeted allele (indicated as -/- or Cc2d2a^-/-) lacked the Cc2d2a transcript (Fig. 1c). Finally, we demonstrated by immunoblot analysis that Cc2d2a^-/- embryos lacked the CC2D2A protein (Fig. 1d, Supplementary Fig. 2).

The analysis of F2 litters produced by crossing heterozygous Cc2d2a^+/-mice identified live pups with only Cc2d2a^+/+ and Cc2d2a^+/- genotypes, suggesting lethality of Cc2d2a^-/- homozygous null embryos. We then assessed at what age Cc2d2a^-/- embryos are lost. We were able to identify Cc2d2a^-/- embryos at close to predicted ratios (25%) on embryonic day (E) 14 and E16. However, the ratio of Cc2d2a^-/- embryos declined sharply at E18 (to 4%), and null mutants rarely survived beyond that age (Table 1). At E16-18, Cc2d2a^-/- embryos displayed pleiotropic phenotypes resembling MKS, with many showing severe degeneration and resorption during early embryogenesis (Fig. 1e). In addition to hemorrhage, open neural tube, microphthalmia, and anophthalmia, Cc2d2a^-/- embryos often revealed situs inversus and dextrocardia, and on occasion lacked abdominal organs (Fig. 1f, boxed area). Polydactyly was observed frequently in Cc2d2a^-/- embryos (Fig. 1h, arrows). Thus, Cc2d2a is broadly required for organogenesis in mice. Among hundreds of Cc2d2a^-/- mutants generated in our laboratory, only one mouse survived for 27 days and showed gross underdevelopment with marked lethargy, loss of hair on its dorsal surface, hydrocephalus (Fig. 1g, compare arrows), and retinal dystrophy (Supplementary Fig. 3).

Cc2d2a^-/- embryos have defects in motile and sensory cilia

The situs inversus phenotype suggested defects in the embryonic node and establishment of left-right asymmetry. Scanning electron microscopy of the E8 Cc2d2a^-/- embryos revealed flattening of the node with only a few cilia-like structures (Fig. 2a-c). Immunostaining of the embryonic node using anti-acetylated α–tubulin antibody validated these findings (Fig. 2d). The analysis of other ciliated tissues also showed profound defects in cilia biogenesis. The kinocilia in the cochlea were frequently absent or abnormal (green signal in Fig. 2e, right) and stereociliary bundles deformed (red signal in Fig. 2e, right) in Cc2d2a^-/- embryos. The cilia were also absent or abnormal in Cc2d2a^-/- embryonic liver (green signal in Fig. 2f, right). Perinatal kidney tubules and tracheal epithelium revealed few cilia in Cc2d2a^-/- embryos (green signal in Fig. 2g and h, right panels). By comparison, kidney tubules in wild type embryos had prominent, primary cilia projecting into the lumen (Fig. 2g, left), and tracheal epithelia presented tufts of motile cilia (Fig. 2h, left).

Shh signaling is perturbed in Cc2d2a^-/- embryos

As Cc2d2a^-/- embryos often exhibited exencephaly (see Fig. 1e), we hypothesized that the open neural tube phenotype is due to abnormal cilia biogenesis and perturbed Shh signaling ^22-24. Our analysis of embryonic day (E) 12 embryos revealed the presence of cilia in the neural tube in the wild type (Fig. 3a, upper middle panel) but not in the Cc2d2a^-/- mutants (Fig. 3a, lower middle panel). We noted the presence of basal bodies in the neural tube of both genotypes (Fig. 3a, left panels).

We then investigated a potential defect in patterning of neural tube domain markers due to lack of cilia-mediated Shh signaling. In wild type embryos, Shh signal localizes to the floor plate of neural tube and at the notochord (Fig. 3b). In contrast, Cc2d2a^-/- embryos lack Shh signal at the floor plate (Fig. 3b). In contrast to the wild type embryos showing Nkx2.2 and Nkx6.1 expression (Fig. 3b) at the ventral neural tube, Nkx2.2 signal was found at the floor plate in the Cc2d2a^-/- embryos and Nkx6.1 expressing domain shifted toward the ventral midline (Fig. 3b). Furthermore, the Pax6 domain also expanded ventrally. These data indicate the loss of floor plate cell fate and a reduction in V3 progenitors, which require high levels of Shh for induction. Olig2 signal, marking progenitors of the oligodendrocyte lineage that also depend on Shh signaling for induction, was absent in the Cc2d2a^-/- embryos (Fig. 3b). We note that Pax7, HB9 and Msx2 showed no discernable difference in Cc2d2a^-/- embryos compared to the wild type (Fig. 3b). Thus, the neural tube patterning defect of Cc2d2a^-/- embryos mostly affects ventral cell fates, consistent with a perturbation of Shh signaling. The neural tube patterning defect in this mutant is similar to that of the Mks1 mutant ²⁵.

Cc2d2a^-/- fibroblasts have basal body but not ciliary axoneme

To delineate the underlying defect in cilia biogenesis, we isolated and cultured embryonic fibroblasts (MEFs) from Cc2d2a^-/- mutants. Unlike MEFs from wild type littermates (Fig. 4a, upper panel), Cc2d2a^-/- MEFs did not grow cilia upon serum starvation (Fig. 4a, lower panel). We however noted that ∼10% of Cc2d2a^-/- MEFs escaped this phenotype. Both Cc2d2a^-/- and wild type MEFs showed similar mother centriole staining with γ-tubulin, yet Arl13b labeling was compromised in Cc2d2a^-/- MEFs (Fig. 4a). In addition, microtubule (MT) staining with acetylated α–tubulin showed presence of axoneme in the wild type but not in Cc2d2a^-/- MEFs; nonetheless, cytoplasmic MTs were visible in Cc2d2a^-/- MEFs (Fig. 4c-d). These data suggest that ciliary axoneme biogenesis was not initiated from the mother centriole in the absence of Cc2d2a. The mouse Cc2d2a transgene could rescue the axoneme assembly defect in Cc2d2a^-/- MEFs (Fig. 4b; note that the non-transfected cells in the same field do not have cilia), demonstrating a direct role of CC2D2A in the genesis of cilia from the mother centriole.

The existence of a mother centriole but lack of axoneme suggested that Cc2d2a is needed in early ciliogenic processes. After polarity-guided centriolar migration, the mother centriole docks to the membrane with distal appendages, whereas the anchoring of MT arrays requires SDA ⁶. Even though MT nucleation starts with aster formation at both centrioles, only the mother centriole is able to sustain a stable MT array, a process requiring ninein ^{26, 27}. Immunolabeling with anti-ninein antibody revealed a significant reduction of ninein signal at the mother centriole in Cc2d2a^-/- MEFs (Fig. 4d). It should be noted that ninein is present predominantly at the distal end of the mother centriole, on SDA, while it is also detectable at the proximal ends of both centrioles. Our results thus indicate a basic structural defect in mother centriole.

Distal end components are abnormal in MC of Cc2d2a^-/- MEFs

Immunolabeling of wild type and Cc2d2a^-/- MEFs using rootletin and pericentrin antibodies revealed no significant differences (Supplementary Fig. 4a and Fig. 4c), implying structural integrity of pre-proximal and proximal ends of the two centrioles. Absence of the ciliary axoneme and reduced ninein staining prompted us to examine Odf2, an established marker of SDA ²⁸, which is needed for cilia biogenesis ²⁹. The anti-Odf2 signal at the mother centriole was dramatically reduced in Cc2d2a^-/- MEFs compared to the wild type (Fig. 4e). We then performed immunolabeling against trichoplein, which controls MT anchoring at the mother centriole by binding to Odf2 and ninein ³⁰. Interestingly, trichoplein signal was also significantly reduced at the mother centriole in Cc2d2a^-/- MEFs (Fig. 4f). Our results suggest that recruitment of Odf2 and trichoplein to the SDA is compromised when CC2D2A is not present, and that CC2D2A is needed for SDA assembly.

CC2D2A is required to form subdistal appendages

The loss of cc2d2a in zebrafish photoreceptors resulted in mislocalisation of Rab8 ³¹, which interacts with Odf2 and is needed for cilia biogenesis ³². In Cc2d2a^-/- MEFs, Rab8 staining is significantly reduced compared to the wild type (Fig. 5a). Furthermore, transfection of Rab8a-mCherry in wild type MEFs showed sharp localization at the base of the cilium, whereas the signal was diffuse and cytoplasmic in Cc2d2a^-/- MEFs (Fig. 5b). Notably, immunoblotting demonstrated similar levels of Rab8a protein in the wild type and Cc2d2a^-/- MEFs (Supplementary Fig. 5). These results thus indicate defective vesicle docking or membrane tethering at mother centriole in the absence of CC2D2A.

To further investigate cellular ultrastructure at and near the mother centriole, we took advantage of transmission electron microscopy (TEM). In wild type MEFs, normal ciliary structures were evident with microtubules anchored at SDA and oriented in a regular array from the cytoplasm toward the basal body (Fig. 5c; left, arrow). However, in Cc2d2a^-/- MEFs, mother centriole lacked or had abnormal SDA, had disorganized microtubules and showed accumulation of vesicle-like structures nearby (Fig. 5c, middle panel). Notably, in a fraction of Cc2d2a^-/- MEFs, the mother centriole docked to ciliary vesicles but apparently no transition zone developed (Fig. 5c, right panel). This finding was not obtained in the wild type MEFs. We therefore conclude that CC2D2A is needed for the assembly of SDA at mother centriole, for stable MT anchoring and docking of transport vesicles, and for further elaboration of the transition zone.

MT regrowth assays using α- and γ-tubulin ³³ were then performed to evaluate whether MT anchoring at the mother centriole requires CC2D2A. In wild type and Cc2d2a^-/- MEFs, MT regrowth did not initiate immediately after removing nocodazole (Fig. 6, 0 min). At 5 min, the MT started reappearing at the MC in the wild type, but not seen in Cc2d2a^-/- MEFs. At 30 min of recovery from nocodazole MT aster anchoring at the mother centriole was visible in wild type cells (Fig. 6, 30 min). At 30 min, though MTs were visible in Cc2d2a^-/- MEFs, these did not form an aster and anchor at the basal body. These data demonstrate a critical role of CC2D2A in MT anchoring at the basal body.

CC2D2A localizes to subdistal appendages

Immunolabeling in ciliated IMCD3 cells revealed the localization of CC2D2A protein to the mother centriole. The CC2D2A staining partially overlapped with RPGR, a cilia transition zone marker ³⁴, and anti-GT335 (Fig. 7a, b), which labels polyglutamylated tubulin and marks both the basal body and the proximal axoneme ^{35, 36}. Interestingly, CC2D2A immunostaining appeared similar to that of centriolin ³⁵ and Odf2 ²⁸. To further refine the localization of CC2D2A, we took advantage of immuno-EM. Immunogold labeling for CC2D2A was clearly visible on the SDA both in IMCD3 cells and in monkey retina (Fig. 7c, d). Our data are concordant with preceding MEF studies and strengthen the proposition of a role for CC2D2A in SDA assembly.

No cell cycle defect in Cc2d2a^-/- MEFs

We detected similar anti-GT335 immunostaining in wild type and Cc2d2a^-/- MEFs (Supplementary Fig. 4b, lower panels), consistent with the hypothesis that CC2D2A includes a catalytically inactive transglutaminase like domain ¹⁹. To test whether CC2D2A might be required for sensing Ca²⁺ level changes during cell cycle ³⁷, we analysed the cell cycle profile in MEFs. No significant difference was identified between the wild type and Cc2d2a^-/- MEFs (Supplementary Fig. 6).

Generation of Cc2d2a^-/- mice and MEFs for phenotyping

All animal experiments were performed after obtaining approval of the animal care and use committee of the National Eye Institute (ASP-NEI 650).

The mouse Cc2d2a gene on chromosome 5 spans almost 80 kb and includes 40 exons (Gene ID: 231214) with three protein-coding splice variants. Following standard homologous recombination ^{41, 57}, Exons 6 to 8 were replaced with a gene-targeting cassette containing homology arms and a β-gal reporter. Southern blotting and PCR validated the correct integration of the targeting cassette. qRT-PCR analysis of different tissues was performed using a TaqMan assay (Applied Biosystems; Mm01211431_m1). The method for RNA-seq analysis of MEFs has been reported ⁵⁸. The procedures for electroretinography (ERG), histology and immunohistochemistry are reported elsewhere ⁵⁹. Analysis of the neural tube for cilia and Shh signaling associated proteins was largely performed as detailed ⁴³.

MEFs were prepared from E12.5-E13.5 mouse embryos, as instructed (Millipore Tech. Publications). For immunostaining, 70,000 MEFs were plated in each well of a chamber slide (ibidi, WI) with 300 μl of medium. After overnight growth, cilia biogenesis was induced with serum free medium. The cells were grown to confluence for 48 h, washed with PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, and subjected to standard immunostaining protocol. MEFs were imaged on LSM 700 (Zeiss, Germany) confocal microscope, and the images were edited with Adobe Photoshop CS5 and assembled on Adobe Illustrator CS5. The fluorescence intensity in the images was analyzed with ImageJ64 (NIH) as performed earlier ⁶⁰. Briefly, fluorescence signal (pixel area) in an image was selected with a tool (circle) and integrated density (mean gray value) of the area was measured. Similarly, from the same field, an area with no fluorescence signal (next to a cell) was measured for background intensity. The corrected fluorescence (CF) was calculated by the formula, CF= integrated density – (area of pixel with signal X mean fluorescence of background readings). Statistical analysis of difference in fluorescence intensity was performed by t-test (2 tailed, type 2) in Excel (Microsoft).

Antibodies

The following primary antibodies were used: anti-CC2D2A (Rb, 1:300, custom made), anti-acetylated α-tubulin (Ms, 1:500; Sigma, T6793), anti-α-tubulin (Ms, 1:2000; Sigma, T6199 (DM1A)), anti-γ–tubulin (Ms, 1:500; Sigma, T6557), anti-GT335 (Ms, 1:500; Adipogen, AG-20B-0020), anti-Arl13b (Rb, 1:500; Proteintech, 17711-1-AP;), anti-trichoplein (Rb, 1:100; Masaki Inagaki), anti-human cenexin (ODF2 Rb, 1:50; Kyung Lee), anti-ninein-L77 and –L79 (Rb, 1:2500; Michel Bornens), anti-Rab8a (Rb, 1:250; Proteintech, 55296-1-AP), anti-rootletin6 (Rb, 1:500; Tiansen Li) and anti-RPGR-S3 (Chk, 1:300; Tiansen Li), anti-pericentrin (Rb, 1:500; Abcam, ab448), Anti-Olig2 (Rb, 1:200; R&D, AF2418). The following monoclonal antibodies (Ms, 1:200, DSHB): Shh, Nkx2.2, Nkx6.1 (clone F55A10), HB9, Msx2, Islet-1 (clone 40.2.D6) Pax6 and Pax7. The secondary antibodies for immune-fluorescence analysis were coupled to Alexa Fluor 488 or 568 dyes (Molecular Probes).

Microtubule regrowth assay

MEFs were plated at a high confluence onto the chamber slide (70-80%) and the following day the cells were treated with 5 uM nocodazole (Sigma) in complete media (DMEM) and incubated for 1.5 h at 37°C. Subsequently, the media was aspirated and the cells were washed once with PBS and replaced with complete media. After treatment, the cells were fixed at time points of interest with 100% methanol 3 min on ice and then processed for immunofluorescence staining, with anti-γ–tubulin and anti-α-tubulin, sequentially. The images were captured on LSM 700 confocal microscope (Zeiss, Germany).

Scanning Electron Microscopy

Embryos (E8) were fixed in paraformaldehyde (4%) and glutaraldehyde (2%) in cacodylate buffer (0.1 M) for 2 h at room temperature and then washed in cacodylate buffer thrice (10 min each). The embryos were treated for 1 h in osmium tetroxide (1%), washed thrice (10 min each) in cacodylate buffer, and dehydrated in graded ethanol (35%, 50%, 70%, 95%, 2 times for 10 min each, and then 3 times with 100%). After treating with tetramethylsilane solution (3 times for 10 min each), the embryos were mounted on a SEM stub and sputter coated with gold/palladium using an EMITECH K575 high-resolution coater. Imaging was performed on S-3000N scanning electron microscope (Hitachi, Japan).

Transmission Electron Microscopy (TEM) and Immuno EM

The serum-starved MEFs were fixed and processed for TEM as described ⁶¹. Sections of cells in Epoxy blocks (80 nm) were prepared with Leica UCT Ultramicrotome, mounted on 200 mesh copper grids and coated with carbon. The sections were imaged using H-7600 transmission electron microscope (Hitachi).

Post embedding Immuno EM

The procedure of post-embedding immuno EM was previously described ⁶². Briefly, cultured IMCD3 cells (CRL-2123, ATCC) were fixed in phosphate-buffered saline containing formaldehyde (4% v/v) and glutaraldehyde (0.05% v/v) for 2 h, then dehydrated in a series of cold ethanol (35%, 50%, 70%, 95%, and 100%). The cells were infiltrated in 1:1 and 1:2 mixtures of 100% ethanol and LR White resin for 1hr each, then in LR White resin overnight. The cells were embedded in LR White resin and cured in a 55°C oven for 24 h. Thin sections (90-100 nm) were then mounted on 300-meshed nickel grids that were first blocked by a commercial blocking buffer for goat antibodies and then incubated in a serial dilution of primary antibody, followed by immunogold-conjugated secondary antibody (10 nm particles). The grids were washed in Tris-buffer (pH 7.4) containing BSA (0.1% w/v), NaCl (250 mM), and Tween-20 (0.01% v/v). The grids were stained in uranyl acetate and lead citrate and examined by electron microscope.

Post embedding immunolabeling of monkey photoreceptors

Monkey eyes (Macaca mulatta) were obtained from the Washington National Primate Research Center at the University of Washington. Eyes were fixed in 2% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer for 1 h, then en bloc stained in uranyl acetate, dehydrated and infiltrated in LR White as previously described ⁶³. Thin sections were collected on nickel grids, blocked in normal goat serum and incubated in anti-CC2D2A antibody (1:100) for 2 h, washed and incubated in goat anti-rabbit 10 nm gold secondary for 1 h. Sections were stained with uranyl acetate and lead citrate and examined on a JEOL 1010 transmission electron microscope. Negative controls (no primary) were run in parallel.

Flow cytometry

DNA content was assayed by flow cytometry using propidium iodide. Samples were acquired using a FACSCalibur (BD Bioscience, CA) and the data analysed with FlowJo V9.5 (TreeStar Inc, OR) using Watson Pragmatic Modeling.